JP2015038059A - Treatment of eye diseases and excessive neovascularization using combined therapy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for treating or preventing eye diseases caused by excessive neovascularization by vascular endothelial growth factor (VEGF)-signaling.SOLUTION: The pharmaceutical composition is i) mesenchymal precursor cells, and ii) a compound that disrupts vascular endothelial growth factor (VEGF)-signaling, where the compound is either: a polypeptide which is an antibody, an antibody-related molecule, and/or a fragment of an antibody or an antibody-related molecule; or a polynucleotide which is, or encodes one or more of, an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, or a duplex RNA molecule.

Description

  The present invention relates to methods of treating or preventing ocular diseases and angiogenesis-related diseases by combination therapy comprising administration of compounds that disrupt cells and VEGF signaling.

Angiogenesis Angiogenesis (or neovascularization) is the formation and differentiation of new blood vessels. Angiogenesis is generally not present in healthy adults or mature tissues. However, angiogenesis occurs in a healthy body to heal wounds and to restore blood flow to tissues after injury or injury. In women, angiogenesis also occurs during the menstrual cycle and during pregnancy. Under these processes, the formation of new blood vessels is tightly regulated.

Angiogenesis and disease In many severe disease states, the body loses control over angiogenesis. Excessive angiogenesis occurs in diseases such as cancer, macular degeneration, diabetic retinopathy, arthritis and psoriasis. In these conditions, new blood vessels nourish the diseased tissue, destroy normal tissue, and in the case of cancer, new blood vessels leak tumor cells into the circulation and remain in other organs ( Tumor metastasis).

  The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971 (Folkman, 1971). Simply put, the hypothesis suggests that tumor volume expansion beyond a certain stage requires the induction of new capillaries. For example, early prevascular stage lung micrometastasis in mice is undetectable except by high power microscopy on tissue sections. Furthermore, indirect evidence supporting the notion that tumor growth is angiogenesis-dependent is described in US Pat. Nos. 5,639,725, 5,629,327, 5,792,845, No. 5,733,876 and 5,854,205.

  To stimulate angiogenesis, tumors contain their own fibroblast growth factors (αFGF and βFGF) (Kandel et al., 1991) and vascular endothelial growth factor / vascular permeability factor (VEGF / VPF) and HGF Upregulates the production of various angiogenic factors. However, many malignancies also produce angiogenesis inhibitors including angiostatin protein and thrombospondin (Chen et al., 1995; Good et al., 1990; O'Reilly et al., 1994). The angiogenic phenotype is believed to be the result of a final equilibrium between these positive neovascularization and negative regulators (Good et al., 1990; O'Reilly et al. , 1994). Several other endogenous inhibitors of angiogenesis have also been identified, but not all are related to the presence of a tumor. These are induced by platelet factor 4 (Gupta et al., 1995; Maione et al., 1990), interferon-α, interleukin-12 and / or interferon-γ (Voest et al., 1995). Inducible protein 10 (Angiolillo et al., 1995; Strieter et al., 1995), gro-β (Cao et al., 1995) and a 16 kDa N-terminal fragment of prolactin (Clapp et al., 1993).

Eye diseases Many eye diseases or disorders caused by dysfunction of eye tissues or structures can lead to decreased vision or complete loss of vision. Ophthalmological diseases, such as television, computers, game consoles and other digital devices, and diseases including dry eye and eye strain due to extensive use of contact lenses have increased in recent years.

  Of the eye diseases, age-related macular degeneration (AMD) is particularly common in the elderly population of Western societies. AMD is the most common cause of legal irreversible blindness in patients over the age of 65 and in the United States, Canada, England, Wales, Scotland and Australia. The average age of patients when they lose central vision in the first eye is about 65 years, but some patients develop signs of the disease in their 40s or 50s. The number of people affected by this disease is steadily increasing due to our modern lifestyle and the extension of life expectancy.

  Neovascularization in the eye is fundamental to severe eye diseases such as AMD and diabetic retinopathy. About 10% to 15% of patients develop this disease in the wet form. Wet AMD is characterized by the formation of angiogenesis and pathological neovasculature. The disease is bilateral and the likelihood of developing a disorder that leads to blindness in the other eye accumulates approximately 10% to 15% every year.

  Diabetic retinopathy is a complication of diabetes that occurs in about 40-45% of patients diagnosed with either type I diabetes or type II diabetes. Diabetic retinopathy usually occurs in both eyes and progresses over four stages. Stage 1 mild nonproliferative retinopathy is characterized by an intraocular microaneurysm. Small areas of swelling occur in the capillaries and small blood vessels of the retina. In stage 2 moderate nonproliferative retinopathy, the blood vessels that supply the retina become occluded. In stage 3, severe non-proliferative retinopathy, the occluded blood vessels lead to a decrease in blood supply to the retina so that the retina generates new blood vessels to provide blood supply to the retina (angiogenesis) Signals to the eye. In the most advanced stage 4 proliferative retinopathy, angiogenesis occurs, but new blood vessels are abnormal, fragile, and grow along the surface of the vitreous gel filling the retina and eyes. If these thin blood vessels rupture or leak blood, severe vision loss or blindness can occur.

  Bevacizumab is a compound that has been used to treat AMD, but the side effect of this treatment is increased retinal detachment (Chan et al., 2007; Kook et al., 2008; Garg et al., 2008 ).

  With aging, the vitreous humor changes from a gel to a liquid, with which it gradually contracts and separates from the retinal ILM. This process is known as “posterior vitreous detachment” (PVD) and occurs normally after age 40. However, degenerative changes in the vitreous can also be induced by pathological conditions such as diabetes, Eales disease and uveitis. PVD can also occur earlier than normal in people with myopia and with cataract surgery. Usually, the vitreous makes a clean break from the retina. Sometimes, however, the vitreous adheres closely to the retina at a specific location. These small lesions of the vitreous resistant and abnormally tight bond can transmit large traction forces from the vitreous to the retina at the binding site. This continuous traction by the vitreous often results in horseshoe-shaped lacerations in the retina. If the retinal laceration is not repaired, the vitreous humor can exude into the retina or into the lower part of the retina and cause retinal detachment, a condition that threatens very severe vision. In addition, the persistent bond between the vitreous and the ILM can cause bleeding due to rupture of blood vessels, which results in vitreous opacification and opacification.

  Incomplete PVD development affects many vitreous retinal diseases, including vitreous macular traction syndrome, vitreous hemorrhage, macular hole, macular edema, diabetic retinopathy, diabetic macular disease and retinal detachment .

US Pat. No. 5,639,725 US Pat. No. 5,629,327 US Pat. No. 5,792,845 US Pat. No. 5,733,876 US Pat. No. 5,854,205

Folkman, 1971 Kandel et al., 1991 Chen et al., 1995 Good et al., 1990 O'Reilly et al., 1994 Good et al., 1990 O'Reilly et al., 1994 Gupta et al., 1995 Maione et al., 1990 Voest et al., 1995 Angiolillo et al., 1995 Strieter et al., 1995 Cao et al., 1995 Clapp et al., 1993

  There is a need for additional therapies that can be used to treat or prevent eye diseases and / or angiogenesis related disorders.

  The inventors have surprisingly found that combination therapies comprising compounds that disrupt cells and VEGF signaling are synergistic when used to treat or prevent eye diseases. . Thus, in a first aspect, the present invention provides a method for treating or preventing an eye disease in a subject comprising administering i) a cell and ii) a compound that disrupts vascular endothelial growth factor (VEGF) signaling. Providing a method comprising:

  Examples of ocular diseases that can be treated or prevented using the methods of the present invention are retinal ischemia, retinal inflammation, retinal edema, retinal detachment, macular hole, traction retinopathy, vitreous hemorrhage, traction macular disease, diabetic Retinopathy, diabetic macular edema, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, post-lens fibroproliferation and / or rubeosis are not limited to these. In a preferred embodiment, the eye disease is retinal detachment, diabetic retinopathy, retinopathy of prematurity and / or macular degeneration.

  In one embodiment, the macular degeneration is atrophic age-related macular degeneration or wet age-related macular degeneration. Preferably, the macular degeneration is wet age-related macular degeneration.

  Previously, applicants have shown that stem cells or their progeny can be used to treat or prevent angiogenesis related disorders (see WO 2008/006168). Applicants have also surprisingly found that combination therapies comprising compounds that disrupt cells and VEGF signaling are synergistic when used to treat or prevent angiogenesis-related disorders. It was. Thus, in a second aspect, the present invention provides a method for treating or preventing an angiogenesis-related disease in a subject, comprising i) a cell, and ii) a compound that disrupts vascular endothelial growth factor (VEGF) signaling. A method comprising administering to a subject is provided.

  Examples of angiogenesis-related diseases that can be treated or prevented using the methods of the present invention include angiogenesis-dependent cancers, benign tumors, rheumatoid arthritis, psoriasis, ocular neovascular diseases, Osler-Weber syndrome, myocardial neovascularization, plaque neovascularization Formation, telangiectasia, hemophilic arthropathy, angiofibroma, wound granulation, intestinal adhesion, atherosclerosis, scleroderma, hyperplastic scar, cat scratch disease and Helicobacter pylori Including but not limited to ulcers.

  In one embodiment, the cell is a stem cell or a progeny cell thereof. In a preferred embodiment, the stem cells are obtained from bone marrow or eye.

Preferably, the stem cell is a mesenchymal progenitor cell (MPC). Preferably, the mesenchymal progenitor cells are TNAP ⁺ , STRO-1 ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , CD45 ⁺ , CD146 ⁺ , 3G5 ⁺ or any combination thereof. In another embodiment, at least some of the STRO-1 ⁺ cells are STRO-1 ^bri .

In a further embodiment, the MPC is not grown in culture and is TNAP ⁺ .

  In a preferred embodiment, the progeny cells are obtained by culturing MPCs in vitro.

  In one embodiment, the compound binds to and / or reduces the production of vascular endothelial growth factor. Preferably, the vascular endothelial growth factor is VEGF-A, VEGF-B, VEGF-C and / or VEGF-D. More preferably, the vascular endothelial growth factor is VEGF-A.

  In one embodiment, the compound that decreases the production of vascular endothelial growth factor binds to hypoxia-inducible factor 1 (HIF-1) and / or reduces the production of hypoxia-inducible factor 1.

  In an alternative embodiment, the compound binds to vascular endothelial growth factor receptor and / or reduces production of vascular endothelial growth factor receptor. Preferably, the vascular endothelial growth factor receptor is selected from VEGFR1, VEGFR2 and / or VEGFR3. More preferably, the vascular endothelial growth factor receptor is VEGFR1 and / or VEGFR2.

  In yet another alternative embodiment, the compound binds to a molecule involved in intracellular signaling induced by vascular endothelial growth factor receptor, eg, vascular endothelial growth factor that binds to VEGFR tyrosine kinase, and / or Reduce the production of the molecule.

  In one embodiment, the compound is a polypeptide. More preferably, the polypeptide is an antibody, an antibody-related molecule, and / or a fragment of any one thereof.

  In another embodiment, the compound is a polynucleotide. Examples include, but are not limited to, antisense polynucleotides, sense polynucleotides, catalytic polynucleotides, double stranded RNA molecules, or polynucleotides that encode any one or more thereof.

  In one embodiment, at least some of the cells are genetically modified.

  Also provided is the use of a compound that disrupts cellular and VEGF signaling for the manufacture of a medicament for use in a combination therapy for treating or preventing an eye disease in a subject.

  Further provided is the use of a compound that disrupts cellular and VEGF signaling as an agent for use in combination therapy to treat or prevent ocular disease in a subject.

  Also provided is the use of a compound that disrupts cellular and VEGF signaling for the manufacture of a medicament for use in a combination therapy for treating or preventing an angiogenesis-related disorder in a subject.

  Further provided is the use of a compound that disrupts cellular and VEGF signaling as an agent for use in combination therapy to treat or prevent an angiogenesis related disorder in a subject.

  In a further aspect, the present invention provides a composition comprising a compound that disrupts cell and VEGF signaling, and optionally a pharmaceutically acceptable carrier.

  In another aspect, the present invention provides a kit comprising a cell and a compound that disrupts VEGF signaling. The cells and compounds can be in the same or different containers.

  As will be apparent, the preferred features and characteristics of one aspect of the invention apply to many other aspects of the invention.

  Throughout this specification the word “comprise” or variations such as “comprises” or “comprising” mean inclusion of the stated elements, integers or steps, or groups of elements, integers or steps. It is understood that this does not mean the exclusion of any other element, integer or step, or group of elements, integers or steps.

Test plan. Allogeneic MPC is equivalent to anti-VEGF in reducing vascular leakage and is synergistic with anti-VEGF. The combination of anti-VEGF and allogeneic MPC removes severe leaking blood vessels. Synergistic effect of the combination of allogeneic MPC and anti-VEGF on highly leaking blood vessels. Continuous prevention of stage 4 disease by combined use of allogeneic MPC and anti-VEGF. However, anti-VEGF alone has only a short-lived effect. The combination of anti-VEGF and allogeneic MPC maintains a higher proportion of laser-damaged blood vessels in stage 1 disease. The combination of anti-VEGF and allogeneic MPC prevents retinal detachment. The combination of anti-VEGF and allogeneic MPC prevents retinal detachment after laser-induced neovascularization.

Key points of the sequence listing SEQ ID NO: 1-human VEGF-A (active processed peptide).
SEQ ID NO: 2-human VEGF-B (active processed peptide).
SEQ ID NO: 3-human VEGF-C (active processed peptide).
SEQ ID NO: 4-human VEGF-D (active processed peptide).
SEQ ID NO: 5-human VEGFR-1 (minus signal sequence).
SEQ ID NO: 6-human VEGFR-2 (minus signal sequence).
SEQ ID NO: 7-human VEGFR-3 (minus signal sequence).
SEQ ID NO: 8-human HIF-1α.
SEQ ID NO: 9—Coding sequence for full length human VEGF-A.
SEQ ID NO: 10—Coding sequence for full length human VEGF-B.
SEQ ID NO: 11—coding sequence for full length human VEGF-C.
SEQ ID NO: 12—Coding sequence for full length human VEGF-D.
SEQ ID NO: 13—Coding sequence for full length human VEGFR-1.
SEQ ID NO: 14—Coding sequence for full length human VEGFR-2.
SEQ ID NO: 15—coding sequence for full length human VEGFR-3.
SEQ ID NO: 16—Coding sequence for human HIF-1α.

General Techniques Unless otherwise stated, all technical and scientific terms used herein are those of ordinary skill in the art (eg, stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry and biological It is interpreted to have the same meaning as commonly understood by one skilled in the art of chemistry).

  Unless otherwise indicated, the recombinant proteins, cell culture, and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. Such techniques are described throughout the literature, e.g., J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. (1989), TA Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all revisions to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all revisions to date) Described and explained.

Treatment or Prevention of Disease As used herein, the term “subject” (also referred to herein as “patient”) includes warm-blooded animals, preferably mammals including humans. Subjects can be, for example, livestock (eg sheep, cows, horses, donkeys, pigs), pet animals (eg dogs, cats), laboratory animals (eg mice, rabbits, rats, guinea pigs, hamsters), or captive wild animals (eg foxes, Deer). In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human.

  As used herein, the terms “treating”, “treat”, or “treatment” are used to reduce or eliminate at least one symptom of an ocular disease and / or angiogenesis-related disorder. Administration of a sufficient therapeutically effective amount of a cell as defined herein and a therapeutically effective amount of a compound as defined herein. In one embodiment, the disease is wet age-related macular degeneration, and the methods of the invention reduce the severity of the disease and / or delay or prevent the recurrence of the disease. In another embodiment, the methods of the invention have a longer duration of action than administration of a compound alone that disrupts vascular endothelial growth factor (VEGF) signaling.

  As used herein, the terms “preventing”, “preventing” or “prevention” are used to prevent or prevent the development of at least one symptom of an ocular disease and / or angiogenesis-related disorder. Administration of a sufficient therapeutically effective amount of a cell as defined herein and a therapeutically effective amount of a compound as defined herein.

Ocular Disease As used herein, an “eye disease” is a disease, illness or condition that affects or involves the eye or one of the parts or regions of the eye. The eye includes the eyeball and the tissues and fluids that make up the eyeball, the periocular muscles (eg, oblique and rectus muscles) and the portion of the optic nerve that is within or adjacent to the eyeball.

  In one embodiment, the eye disease is characterized, at least in part, by retinal detachment and / or vascular leakage.

  It is understood that the methods of the invention can be used to prevent or treat any eye disease or any disease associated with the eye, or in one embodiment, any eye disorder. Examples of ocular diseases that can be treated or prevented using the methods of the present invention are suprasclerosis, scleritis, diabetic retinopathy, glaucoma, macular degeneration, retinal detachment, 1 color vision / Maskun, amblyopia, refractive left / right asymmetry , Argyle Robertson pupil, astigmatism, refraction left-right disorder, blindness, chalazion, color blindness, 1 color vision / Maskun, esotropia, esotropia, floater, vitreous detachment, Fuchs Dystrophy, hyperopia, hypertension Retinopathy, iritis, keratoconus, Leber's congenital cataract, Leber's hereditary optic neuropathy, macular edema, myopia, night blindness, progressive extraocular and internal ocular paralysis, ocular myopathy Presbyopia, pterygium, red eye (medicine), retinitis pigmentosa, retinopathy of prematurity, retinolysis, river blindness, eye muscle paralysis, dark spot, snow blindness / arc eye , Eyelid disorders, drooping , Extraocular tumors, including perspective, but are not limited to.

  In one preferred embodiment, the methods of the invention can be used to prevent or treat macular degeneration. In one embodiment, macular degeneration is characterized by damage to the macula or destruction of the macula, which in one embodiment is a small area on the back of the eye. In one embodiment, macular degeneration causes progressive loss of central vision but does not lead to complete blindness. In one embodiment, macular degeneration is atrophic, and in another embodiment, macular degeneration is wet. In one embodiment, the contracted form is characterized by macular tissue thinning and loss of function. In one embodiment, the wet type is characterized by the growth of abnormal blood vessels behind the macula. In one embodiment, abnormal blood vessels bleed or leak and cause the formation of scar tissue if not treated. In some embodiments, atrophic macular degeneration may change to wet type. In one embodiment, macular degeneration is age-related, which in one embodiment is through choriocapillary ingrowth through Bruch's membrane defect with growth of fibrovascular tissue beneath the retinal pigment epithelium. Is caused.

  In another preferred embodiment, the methods of the invention can be used to prevent or treat retinopathy. In one embodiment, retinopathy refers to a retinal disease, which in one embodiment is characterized by inflammation, and in another embodiment, due to vascular damage within the eye. In one embodiment, the retinopathy is diabetic retinopathy, which in one embodiment is a diabetic complication caused by changes in retinal blood vessels. In one embodiment, blood vessels in the retina leak blood and / or grow fragile brush-like branches and scar tissue, which in one embodiment blurs the image that the retina sends to the brain. Or deform. In another embodiment, the retinopathy is proliferative retinopathy, which in one embodiment is characterized by the growth of new abnormal blood vessels on the surface of the retina (neovascularization). In one embodiment, neovascularization around the pupil increases intraocular pressure, which in one embodiment leads to glaucoma. In another embodiment, neovascularization leads to new blood vessels with fragile walls that break and bleed or grow scar tissue, which in one embodiment pulls the retina away from the back of the eye (retina Peeling). In one embodiment, the etiology of retinopathy is non-enzymatic saccharification reaction, glycation reaction, accumulation of end product of advanced glycation reaction, free radical mediated protein damage, matrix metalloproteinase upregulation, growth factor refinement Related to elaboration, secretion of adhesion molecules in the vascular endothelium, or combinations thereof.

  In another preferred embodiment, retinopathy refers to retinopathy of prematurity (ROP), which in one embodiment occurs in premature infants when abnormal blood vessels and scar tissue grow on the retina. In one embodiment, retinopathy of prematurity is caused by the treatment necessary to promote the survival of premature infants.

  In another preferred embodiment, the methods of the invention can be used to prevent or treat retinal detachment, including, among others, hiatogenic, traction or exudative retinal detachment, In an embodiment, the separation of the retina from its support layer. In one embodiment, retinal detachment is associated with a tear or hole in the retina through which intraocular fluid can leak. In one embodiment, retinal detachment is caused by trauma, aging processes, severe diabetes, inflammatory disorders, neovascularization, or retinopathy of prematurity, and in another embodiment occurs spontaneously. In one embodiment, bleeding from small retinal vessels can turbid the vitreous during detachment, which in one embodiment can produce a blurred and deformed image. In one embodiment, retinal detachment can cause severe vision loss including blindness.

Angiogenesis As used herein, the term “angiogenesis” is defined as the process of tissue angiogenesis involving the growth of new and / or developing blood vessels into tissue, also referred to as neovascularization. Is done. This process can be performed in any of three ways: a vessel can sprouting from an existing vessel, a new development of a vessel can arise from progenitor cells (angiogenesis), and / or the diameter of an existing small vessel can be expanded. It can go on.

  As used herein, an “angiogenesis-related disease” is any condition characterized by excessive and / or abnormal neovascularization. Any angiogenesis-related disease can be treated or prevented using the methods of the present invention. Angiogenesis-related diseases are angiogenesis-dependent cancers such as solid tumors, blood-borne tumors such as leukemia and tumor metastasis; benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachoma and pyogenic granulomas; rheumatoid arthritis Psoriasis; ocular neovascular diseases such as diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, macular degeneration including aged age-related macular degeneration and wet age-related macular degeneration, corneal graft rejection, angiogenesis Glaucoma, post-lens fibroproliferation, lebeosis; Osler-Weber syndrome; myocardial angiogenesis blindness; plaque neovascularization; telangiectasia; hemophilic arthropathy; angiofibroma; and wound granulation Including, but not limited to. The methods of the present invention are also useful in the treatment or prevention of diseases that have angiogenesis as a pathological consequence, such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylorii).

  In a preferred embodiment, the angiogenesis related disease is an ocular neovascular disease. As used herein, an “ocular neovascular disorder” is any ocular disorder characterized by excessive and / or abnormal neovascularization. Examples include, but are not limited to, diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, post-lens fibroproliferation, and rubeosis.

Stem cells and their progeny cells The cells can be any cell type that can be used to treat ocular diseases and / or angiogenesis related disorders.

  As used herein, the term “stem cell” can give rise to a phenotypically and genotypically identical daughter cell and at least one other final cell type (eg, a terminally differentiated cell). Can refer to self-renewable cells. The term “stem cell” encompasses totipotent, pluripotential and multipotential cells, as well as progenitor and / or progenitor cells derived from their differentiation.

  As used herein, the term “totipotent cell” or “totipotent cell” refers to a cell that can form a complete embryo (eg, a blastocyst).

  As used herein, the term “pluripotent cell” or “pluripotent cell” refers to full differentiation diversity, the ability to grow into any of the approximately 260 cell types of the mammalian body. Refers to the cells that have them. Pluripotent cells can be self-renewing and can remain dormant or quiescent within the tissue.

  By “multipotent cell” or “multipotent cell” is meant a cell that can give rise to any of several mature cell types. As used herein, this phrase includes adult or embryonic stem cells and progenitor cells, such as mesenchymal progenitor cells (MPC) and pluripotent progeny of these cells. Unlike pluripotent cells, pluripotent cells do not have the ability to form all cell types.

  As used herein, the term “progenitor cell” refers to a cell that has been determined to differentiate into a specific cell type or to form a specific tissue type.

  Mesenchymal progenitor cells (MPC) are bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, eye including retina, brain, hair follicle, intestine, lung, lymph node , Cells found in thymus, bone, ligament, tendon, skeletal muscle, dermis, and periosteum, and can differentiate into various germ cell lines such as mesoderm, endoderm and ectoderm. Thus, MPC can differentiate into a number of cell types including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage determination and differentiation pathways into which these cells enter can vary from mechanical effects and / or local microenvironmental conditions established by endogenous bioactive factors such as growth factors, cytokines, and / or host tissues. Depends on the impact. Mesenchymal progenitor cells are therefore non-hematopoietic progenitor cells that divide to give rise to daughter cells that are either stem cells or progenitor cells that eventually differentiate irreversibly into one phenotype. Give rise to cells.

  In a preferred embodiment, the cells used in the methods of the invention are enriched from a sample obtained from a subject. The terms “enriched”, “enriched” or variations thereof refer to a population of cells in which the ratio of one specific cell type or the ratio of multiple specific cell types is increased compared to an untreated population. Used herein to represent.

In a preferred embodiment, the cells used in the present invention are TNAP ⁺ , STRO-1 ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , CD45 ⁺ , CD146 ⁺ , 3G5 ⁺ or any of them It is a combination. Preferably, the STRO-1 ⁺ cells are STRO-1 ^bright . Preferably, the STRO-1 ^bright cells are further one or more of VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ and / or CD146 ⁺ .

  In one embodiment, the mesenchymal progenitor cells are perivascular mesenchymal progenitor cells as defined in WO 2004/85630.

  When we refer to a cell as “positive” for a given marker, it depends on the degree to which the marker is present on the cell surface, depending on the low (lo or dim) expression of that marker. It can be either a factor or a high (bright or bri) expression factor, in which case the term relates to the fluorescence or other color intensity used in the cell color sorting process. The distinction between lo (or dark or dull) and bri is understood in relation to the markers used for the particular cell population being sorted. When we refer to a cell as “negative” for a given marker herein, it does not mean that the marker is not expressed at all by that cell. That means that the marker is expressed at a relatively very low level by the cell and produces a very low signal when detectably labeled.

The term “bright” as used herein refers to a cell surface marker that produces a relatively high signal when detectably labeled. Without wishing to be bound by theory, it is proposed that “bright” cells express more target marker protein (eg, an antigen recognized by STRO-1) than other cells in the sample. For example, STRO-1 ^bri cells produce a greater fluorescent signal than non-bright cells (STRO-1 ^{dull / dark} ) when labeled with a FITC-conjugated STRO-1 antibody as measured by FACS analysis. Preferably, “bright” cells constitute at least about 0.1% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In other embodiments, the “bright” cells are at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1. of the brightest labeled bone marrow mononuclear cells contained in the starting sample. 5%, or at least about 2%. In a preferred embodiment, STRO-1 ^bright cells have 2 log magnitude higher expression of STRO-1 surface expression. This "background", i.e. STRO-1 ^- is calculated relative to a be a cell. By comparison, STRO-1 ^dark and / or STRO-1 ^intermediate cells are less than 2 log higher in expression of STRO-1 surface expression, typically about 1 log or less higher than “background” Have expression.

  As used herein, the term “TNAP” is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term includes liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In a preferred embodiment, the TNAP is BAP. In a particularly preferred embodiment, TNAP as used herein is produced by a hybridoma cell line deposited with the ATCC on 19 December 2005 under the deposit accession number PTA-7282 according to the provisions of the Budapest Treaty. Refers to a molecule capable of binding to a STRO-3 antibody.

  Furthermore, in a preferred embodiment, the cells can give rise to clonogenic CFU-F.

  It is preferred that a significant proportion of pluripotent cells can differentiate into at least two different germline. Non-limiting examples of lineages from which pluripotent cells can be differentiated include: osteoprogenitor cells; hepatocyte progenitor cells with multipotency into bile duct epithelial cells and hepatocytes; oligodendrocytes and Neural restricted cells that can produce glial progenitor cells that progress to astrocytes; neural progenitor cells that progress to neurons; myocardial and cardiomyocyte progenitor cells; glucose-responsive insulin-secreting pancreas Includes beta cell lines. Other lineages include odontoblasts, dentin producing cells and chondrocytes, and precursor cells of the following cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal canal Epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendon cells, ligament cells, chondrocytes, adipocytes, fibroblasts, bone marrow stromal cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, Including, but not limited to, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes and oligodendrocytes.

  In one embodiment, the stem cells and their progeny can differentiate into pericytes.

  In another embodiment, “pluripotent cells” cannot give rise to hematopoietic cells after culture.

  Stem cells useful for the methods of the invention can be derived from adult tissue, embryos, or fetuses. The term “adult” is used in its broadest sense and encompasses postnatal subjects. In a preferred embodiment, the term “adult” refers to a subject who is after puberty. As used herein, the term “adult” can also include umbilical cord blood collected from women.

  The present invention also relates to the use of progeny cells (also referred to as proliferating cells) produced from the in vitro culture of stem cells described herein, including the direct progeny of stem cells as well as their progeny and the like. The proliferating cells of the present invention can have a wide variety of phenotypes depending on the culture conditions (including the number and / or type of stimulating factors in the medium), the number of passages and the like. In certain embodiments, progeny cells are obtained after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages from the parent population. However, progeny cells can be obtained after any number of passages from the parent population.

  Progeny cells can be obtained by culturing in any suitable medium. The term “medium”, when used in connection with cell culture, includes components of the environment that surround the cells. The medium can be a solid, liquid, gas, or a mixture of phases and substances. The medium includes a liquid growth medium as well as a liquid medium that does not maintain cell growth. The medium also includes a gelatinous medium such as agar, agarose, gelatin and a collagen substrate. The term “medium” also refers to a substance that is intended for use in cell culture, even if it is not yet in contact with a cell. In other words, the nutrient rich solution prepared for bacterial culture is a medium. Similarly, a powder mixture that becomes suitable for cell culture when mixed with water or other liquids may be referred to as a “powder medium”.

In one embodiment, the progeny cells useful for the methods of the present invention are TNAP ⁺ cells isolated from bone marrow using magnetic beads labeled with STRO-3 antibody and 20 as described above. Obtained by plating in α-MEM supplemented with 2% fetal bovine serum, 2 mM L-glutamine and 100 μm L-ascorbic acid-2-phosphate (Gronthos et al. (1995) for further details on culture conditions). reference).

In one embodiment, such proliferating cells (after at least 5 passages) are TNAP ⁻ , CC9 ⁺ , HLA class I ⁺ , HLA class II ⁻ , CD14 ⁻ , CD19 ⁻ , CD3 ⁻ , CD11a-c ⁻ , It may be CD31 ⁻ , CD86 ⁻ and / or CD80 ⁻ . However, expression of various markers may be different under different culture conditions than those described herein. Also, cells with these phenotypes may dominate in the expanded cell population, but that does not mean that there is no small percentage of cells that do not have this (these) phenotype (eg, small the percentage of proliferating cells CC9 ^- may be). In one preferred embodiment, the proliferating cells of the present invention are still capable of differentiating into different cell types.

In one embodiment, the proliferating cell population used in the methods of the invention comprises cells in which at least 25%, more preferably at least 50% of the cells are CC9 ⁺ .

In another embodiment, the proliferating cell population used in the methods of the invention comprises cells in which at least 40%, more preferably at least 45% of the cells are STRO-1 ⁺ .

In further embodiments, the progeny cell is LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a / CD49b / CD29, CD49c / CD29, CD49d. / CD29, CD29, CD18, CD61, integrin β6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, leptin-R, (STRO-2 = Leptin-R), RANKL, STRO-1 ^bright and a marker selected from the group consisting of CD146 or any combination of these markers may be expressed.

  In one embodiment, the progeny cell is a pluripotent proliferating MPC progeny (MEMP) as defined in WO 2006/032092. Methods for preparing enriched populations of MPC from which offspring can be derived are described in WO 01/04268 and 2004/085630. In an in vitro situation, MPC rarely exists as an absolutely pure formulation and is generally present with other cells that are tissue-specific differentiated cells (TSCC). WO 01/04268 refers to collecting such cells from bone marrow at a purity level of about 0.1% to 90%. The population containing the MPC from which the progeny are derived can be collected directly from tissue origin or can be a population that has already been expanded ex vivo.

For example, the progeny are collected, uncollected, comprising at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 95% of the total cells in the population in which they are present. It can be obtained from a proliferated, substantially purified MPC population. This level can be determined, for example, by selecting for cells that are positive for at least one marker selected from the group consisting of TNAP, STRO-1 ^bright , 3G5 ⁺ , VCAM-1, THY-1, CD146, and STRO-2. Can be achieved.

  The MPC starting population can be any one or more tissue types described in, for example, WO 01/04268 or 2004/085630, ie bone marrow, dental pulp cells, adipose tissue and skin, or possibly More broadly, on adipose tissue, teeth, pulp, skin, liver, kidney, heart, retina, brain, hair follicle, intestine, lung, spleen, lymph node, thymus, pancreas, bone, ligament, bone marrow, tendon and skeletal muscle Can come from.

MEMPS can be distinguished from freshly collected MPCs in that it is positive for the STRO-1 ^bri marker and negative for the alkaline phosphatase (ALP) marker. In contrast, freshly isolated MPCs are positive for both STRO-1 ^bri and ALP. In a preferred embodiment of the invention, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells are STRO-1 ^bri , ALP ^- with a phenotype of. In a further preferred embodiment, MEMPS is positive for one or more of the markers Ki67, CD44 and / or CD49c / CD29, VLA-3, α3β1. In still further preferred embodiments, MEMP does not show TERT activity and / or is negative for the CD18 marker.

  In one embodiment, the cells are harvested from a patient with angiogenesis-related disease, cultured in vitro using standard techniques, and administered to the patient as an autograft or allograft. In an alternative embodiment, one or more cells of an established human cell line are used. In another useful embodiment of the invention, cells from non-human animals (or from another species if the patient is not human) are used.

  The present invention uses cells from any non-human animal species including, but not limited to, non-human primate cells, ungulates, dogs, cats, rabbits, rodents, birds and fish cells. Can be implemented. Primate cells in which the invention may be practiced include, but are not limited to, chimpanzees, baboons, cynomolgus monkeys, and any other New World or Old World monkey cells. The ungulate cells in which the present invention can be practiced include, but are not limited to, bovine, porcine, sheep, goat, horse, buffalo and wild cow cells. Rodent cells that can practice the invention include, but are not limited to, mouse, rat, guinea pig, hamster and gerbil cells. Examples of rabbit species in which the present invention can be practiced include domesticated rabbits, jack rabbits, hares, cottontail rabbits, snowshoe rabbits and pikas. The chicken (Gallus gallus) is an example of an avian species that can practice the present invention.

  Cells useful for the methods of the invention can be stored prior to use. Methods and protocols for preserving and storing eukaryotic cells and particularly mammalian cells are well known in the art (eg, Pollard, JW and Walker, JM (1997) Basic Cell Culture Protocols, Second Edition, Humana Press, Totowa, NJ; See Freshney, RI (2000) Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, NJ). Any method of maintaining the biological activity of isolated stem cells, eg, mesenchymal stem / progenitor cells, or their progeny can be utilized in connection with the present invention. In one preferred embodiment, the cells are maintained and stored by using cryopreservation.

  In one embodiment, the cell is an allogeneic or autologous cell.

  Examples of other cell types that can be used to treat or prevent eye diseases include WO 07/130060 (adult retinal stem cells from extraretinal tissues), US Patent Application No. 200808868 (retinal stem cells). US Patent Application No. 2001031256 (Neuronal Retinal Cells and Porcine Retinal Pigment Epithelial Cells), US Patent Application No. 2006002900 (Retinal Pigment Epithelial Cells), US Patent Application No. 2007248644 (Muller Stem Cells) and US Patent No. 6,162,428 (hNT). Neuron cells), but is not limited to these.

Examples of other cell types that can be used for the methods of the present invention include CD34 ⁺ hematopoietic stem cells, adipose tissue derived cells, STRO-1 ^- marrow derived MPCs, embryonic stem cells, and bone marrow or peripheral blood mononuclear cells. Including, but not limited to.

Cell Sorting Techniques Cells useful for the methods of the present invention can be obtained using a variety of techniques. For example, a number of cell sorting techniques can be used that physically separate cells by reference to properties associated with cell-antibody complexes or labels attached to antibodies. This label may be a magnetic particle or a fluorescent molecule. The antibodies can be spanned to form aggregates of multiple cells that can be separated by density. Alternatively, the antibody may be bound to an immobilized substrate and the desired cells will adhere to it.

In a preferred embodiment, an antibody (or other binding agent) that binds to TNAP ⁺ , STRO-1 ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , 3G5 ⁺ , CD45 ⁺ , CD146 ⁺ is used. To isolate the cells. More preferably, the cells are isolated using an antibody (or other binding agent) that binds to TNAP ⁺ or STRO-1 ⁺ .

  Various methods for separating cells bound to antibodies from unbound cells are known. For example, the cells bound to the cells (or anti-isotype antibodies) can be labeled and then the cells separated by a mechanical cell sorter that detects the presence of the label. Fluorescence activated cell sorters are well known in the art. In one embodiment, the anti-TNAP antibody and / or STRO-1 antibody is bound to a solid support. Various solid supports are known to those skilled in the art and include, but are not limited to, agarose beads, polystyrene beads, hollow fiber membranes, polymers, and plastic petri dishes. Cells to which the antibody is bound can be removed from the cell suspension simply by physically separating the solid support from the cell suspension.

  Superparamagnetic microparticles can be used for cell separation. For example, the microparticles can be coated with anti-TNAP antibody and / or STRO-1 antibody. The antibody labeled superparamagnetic microparticles can then be incubated with a solution containing the cells of interest. The microparticles bind to the surface of the desired stem cells and these cells can then be collected in a magnetic field.

  In another example, a cell sample is physically contacted with, for example, a solid phase-bound anti-TNAP monoclonal antibody and / or an anti-STRO-1 monoclonal antibody. Solid phase binding can include, for example, adsorbing antibodies to plastic, nitrocellulose, or other surfaces. The antibody can also be adsorbed to the walls of the large pores of the hollow fiber membrane (large enough to allow cell flow). Alternatively, the antibody can be covalently bound to the surface of a bead, eg, Pharmacia Sepharose 6 MB macrobead. The exact conditions and duration of incubation of the solid phase-bound antibody and the stem cell-containing suspension will depend on several factors specific to the system used. The selection of appropriate conditions, however, is well within the skill of the art.

  Next, after allowing sufficient time for the stem cells to bind, unbound cells are eluted with physiological buffer or washed away. Unbound cells can be recovered and used for other purposes or discarded after appropriate testing has been performed to ensure that the desired separation has been achieved. The bound cells are then separated from the solid phase by any suitable method, primarily depending on the nature of the solid phase and the antibody. For example, bound cells can be eluted from a plastic petri dish by vigorous agitation. Alternatively, bound cells can be eluted by enzymatic “nicking” or by digesting an enzyme-sensitive “spacer” sequence between the solid phase and the antibody. Spacers attached to agarose beads are commercially available from, for example, Pharmacia.

  The eluted and enriched fraction of cells may then be washed with buffer by centrifugation, and the enriched fraction may be frozen viable for subsequent use by conventional techniques. It can be stored, grown in culture and / or introduced into a patient.

Compounds that disrupt VEGF signaling Compounds for use in the methods of the invention can be any type of molecule that reduces the ability of VEGF to exert its normal biological effects. For example, the compound may bind to VEGF itself, its receptor, or an intracellular signaling protein or transcription factor that is activated and / or synthesized upon activation of the VEGF receptor after binding by VEGF, or Production can be reduced. Thus, as used herein, the term “compound that disrupts VEGF signaling” refers to the amount of VEGF, VEGF receptors or other molecules involved in VEGF signaling, and / or VEGF correspondingly. Refers to a compound that decreases its ability to signal through a receptor and produce an associated downstream biological action, eg, promote cell proliferation and / or division.

  Binding between a compound and its target (eg, VEGF) can be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction results in a non-covalently bound complex, the resulting bond is typically the result of an electrostatic bond, a hydrogen bond, or a hydrophilic / lipophilic interaction. In one embodiment, the compound is a purified and / or recombinant polypeptide. Particularly preferred compounds are purified and / or recombinant antibodies, antibody-related molecules or antigen-binding fragments thereof.

  Although not required, the compound may specifically bind to the target. The phrase “specifically binds” means that under certain conditions, the compound binds to the target and does not bind in significant amounts to other substances, such as other proteins or carbohydrates. For example, in one embodiment, the compound specifically binds to VEGF-A but not to other VEGF. In another embodiment, a compound is “specifically” if there is a difference of 10-fold or more, preferably 25, 50, or 100-fold greater than the binding of the compound to the target when compared to another protein. Is considered to be “join”.

  Examples of compounds useful for the present invention include quinazoline derivative inhibitors of VEGF (US Patent Application Nos. 2007265286, 20030399491 and US Pat. No. 6,809,097), Quercetin (inhibits VEGF) (International Publication) 02/057473), quinazoline derivative inhibitors of VEGFR tyrosine kinase (US Patent Application No. 2007027145), aminobenzoic acid derivative inhibitors of VEGFR tyrosine kinase (US Pat. No. 6,720,424), pyridine derivative inhibitors of VEGFR tyrosine kinase (US Patent Application No. 20030358409), Recentin (Astra Zeneca) (inhibits all three VEGFRs) (WO 07/060402), Sunitinib (Novar) tis) (inhibits all three VEGFRs) (WO 08/031835 and US Pat. No. 6,573,293), Pegaptanib (Macugen ™) (US Pat. No. 6,051, 698), Axitinib (Pfizer) (inhibits all three VEGFRs) (WO 2004/087152), Sorafenib (Bayer Pharmaceuticals) (WO 07/053573), VEGFR-1 binding peptide (US Patent Application No. 20051000963), arginine-rich anti-vascular endothelial growth factor peptide (US Pat. No. 7,291,601) blocking VEGF binding to the receptor, VEGF trap (VEGF-Trap) (Regeneron Pharma) ceuticals) (US Patent Application No. 2005032699), soluble VEGF receptors (US Patent Application No. 2000061364 and Tseng et al., 2002), VEGF-C and VEGF-D peptide mimetic inhibitors (US Patent Application No. 2002065218). ), PAI-1, which blocks the release of VEGF from the VEGF-heparin complex (US Patent Application No. 20040121955), US Patent Application No. 2002068697, WO 02/081520, US Patent Application No. 20060234941, Including, but not limited to, the inhibitors described in US20020586619, as well as further examples outlined below. In a preferred embodiment, the compound is Lucentis, Avastin or VEGF trap.

Examples of Target Molecules In one embodiment, the target molecule of a compound for disrupting VEGF signaling is vascular endothelial growth factor.

  As used herein, the term “vascular endothelial growth factor” or “VEGF” binds to a cell surface tyrosine kinase receptor (VEGF receptor, or VEGFR) to form angiogenesis, angiogenesis and endothelium. Refers to a family of growth factors that stimulate cell growth (see, eg, Breen, 2007).

  As used herein, “VEGF-A” binds to VEGFR-1 and VEGFR-2 receptors, stimulates endothelial cell mitogenesis and cell migration, stimulates MMOP activity, Refers to a member of the VEGF polypeptide growth factor family that increases αvβ3 activity, promotes vascular lumen creation and fenestration, is chemotactic for macrophages and granulocytes, and at the same time is a potent vasodilator (Breen, 2007; Eremina and Quaggin, 2004). Alternatively, spliced transcript variants of VEGF-A have been identified that give rise to a number of different isoforms of VEGF-A. An example of a VEGF-A polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 1, and variants and / or mutants thereof. An example of an open reading frame encoding preproVEGF-A is shown in SEQ ID NO: 9.

  As used herein, the term “VEGF-B” refers to the family of VEGF polypeptide growth factors that bind to the VEGFR-1 receptor and stimulate angiogenesis, endothelial cell mitogenesis and migration. Refers to members (Breen, 2007; Olofsson et al., 1996). Alternatively, spliced transcript variants of VEGF-B have been identified that give rise to several isoforms of VEGF-B. An example of a VEGF-B polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 2, and variants and / or mutants thereof. Furthermore, SEQ ID NO: 10 shows an example of an open reading frame encoding preproVEGF-B.

  As used herein, the term “VEGF-C” refers to a VEGF polypeptide that binds to VEGFR-2 and Flt4 receptors to stimulate endothelial cell mitogenesis and migration and lymphangiogenesis. Refers to members of the growth factor family (Breen, 2007; Su et al., 2007). VEGF-C undergoes complex proteolytic maturation to form several isoforms, and only the fully processed form can bind to and activate its cognate VEGFR-2 receptor. An example of a VEGF-C polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 3, and variants and / or mutants thereof. An example of an open reading frame encoding preproVEGF-C is shown in SEQ ID NO: 11.

  As used herein, the term “VEGF-D” binds to the VEGFR-2 and VEGFR-3 receptors and angiogenesis, lymphangiogenesis, and endothelial cell mitogenesis and migration. Refers to a member of the VEGF polypeptide growth factor family that stimulates VEGF-D undergoes complex proteolytic maturation to form several isoforms, and only the fully processed form can bind to and activate its cognate VEGFR-2 and VEGFR-3 receptors . An example of a VEGF-D polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 4, and variants and / or mutants thereof. An example of an open reading frame encoding preproVEGF-D is shown in SEQ ID NO: 12.

  In one embodiment, the target molecule for disrupting VEGF signaling is a vascular endothelial growth factor receptor.

  As used herein, the term “VEGFR-1” (also known as Flt-1) refers to member 1 of the VEGF tyrosine kinase receptor family located on the cell surface, where VEGFR-1 is 7 Contains one extracellular immunoglobulin-like domain, one transmembrane domain, and an intracellular domain containing tyrosine kinase function to which VEGF-A and VEGF-B bind (Olsson et al., 2006; Cross et al., 2003). After binding of a ligand (eg, VEGF-A), the VEGFR-1 receptor dimerizes and is activated via transphosphorylation to stimulate angiogenesis, angiogenesis and endothelial cell proliferation. An example of a VEGFR-1 polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 5, and variants and / or mutants thereof. An example of an open reading frame encoding VEGFR-1 is shown in SEQ ID NO: 13.

  As used herein, the term “VEGFR-2” (also known as KDR or Flk-1) refers to member 2 of the VEGF tyrosine kinase receptor family located on the cell surface, where VEGFR-2 is , Seven extracellular immunoglobulin-like domains, one transmembrane domain, and an intracellular domain containing tyrosine kinase function to which VEGF-A, VEGF-C and VEGF-D bind (Olsson et al., 2006 Cross et al., 2003). After binding of the ligand, the VEGFR-2 receptor dimerizes and is activated via transphosphorylation to stimulate angiogenesis, angiogenesis and endothelial cell proliferation. An example of a VEGFR-2 polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 6, and variants and / or mutants thereof. An example of an open reading frame encoding VEGFR-2 is shown in SEQ ID NO: 14.

  As used herein, the term “VEGFR-3” (also known as Flt-4) refers to member 3 of the VEGF tyrosine kinase receptor family located on the cell surface, where VEGFR-3 is 7 Contains one extracellular immunoglobulin-like domain, one transmembrane domain, and an intracellular domain containing tyrosine kinase function to which VEGF-C and VEGF-D bind (Olsson et al., 2006; Cross et al., 2003). After ligand binding, the VEGFR-3 receptor dimerizes and is activated via transphosphorylation to mediate lymphangiogenesis. An example of a VEGFR-3 polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 7, and variants and / or mutants thereof. An example of an open reading frame encoding VEGFR-3 is shown in SEQ ID NO: 15.

  In a further embodiment, the target molecule for disrupting VEGF signaling reduces vascular endothelial growth factor production. For example, the target can be hypoxia-inducible factor 1 (HIF-1).

  As used herein, the term “hypoxia-inducible factor 1” (HIF-1) refers to a transcription factor that regulates genes involved in the response to hypoxia. For example, it is known that HIF-1 upregulates VEGF expression in response to hypoxia (Zhang et al., 2007). HIF-1α is an inducible subunit of HIF-1. An example of a HIF-1 polypeptide includes a protein containing the amino acid sequence shown in SEQ ID NO: 8, and variants and / or mutants thereof. An example of an open reading frame encoding HIF-1 is shown in SEQ ID NO: 16.

  Examples of compounds that target HIF-1 include echinomycin (Kong et al., 2005), BDDF-1 (WO 08/004798), S-2-amino-3- [4′-N , N, -bis (2-chloroethyl) amino] phenylpropionic acid N-oxide dihydrochloride (PX-478) (US Patent Application No. 2005049309), ketomin (Kung et al., 2004), 3- (5′- Hydroxymethyl-2′-furyl) -1-benzylindazole (YC-1) (Yeo et al., 2003), 103D5R (Tan et al., 2005), quinocarmycin monocitrate and its Derivatives (Rapisarda et al., 2002), 3- (5′-hydroxymethyl-2′-furyl) -1-benzylindazole (US Patent Application No. 20041987798), and NSC-134754 and SC-643735 (Chau et al., 2005) including, but not limited to.

In a further embodiment, the target molecule for disrupting VEGF signaling is an intracellular signaling protein or transcription factor that is activated and / or synthesized upon VEGF receptor activation after binding by VEGF.
Antibody-Overview

The antibody may be an intact immunoglobulin or as a modified form of various forms, for example, a domain antibody comprising either a V _H or _VL domain, a dimer of heavy chain variable regions (described for camels, VHH). Present as a modified form including, but not limited to, a light chain variable region dimer (VLL), an Fv fragment comprising only the light and heavy chain variable regions, or an Fd fragment comprising the heavy chain variable region and the CH1 domain Can do. ScFv (Bird et al., 1988; Huston et al., 1988) consisting of variable regions of heavy and light chains linked together to form a single chain antibody, as well as oligomers of scFv such as diabodies and triabodies In the term “antibody”. Non-naturally occurring forms of antibodies comprising at least one CDR, more preferably at least one variable domain, are also referred to herein as “antibody-related molecules”. Also encompassed are antibody fragments, eg, Fab, (Fab ′) ₂ and FabFc ₂ fragments, which comprise portions of the variable and constant regions. Also included are CDR-grafted antibody fragments and oligomers of antibody fragments. The heavy and light chain components of the Fv may be derived from the same antibody or from different antibodies, thereby producing a chimeric Fv region. The antibody can be of animal (eg mouse, rabbit or rat) or human origin, or it can be a chimeric (Morrison et al., 1984) or humanized antibody (Jones et al., 1986). As used herein, the term “antibody” encompasses these various forms. In the methods of the present invention, using the guidelines provided herein and methods well known to those skilled in the art as described in the references cited above and published references such as Harlow & Lane (supra). Antibodies for use can be readily produced.

The antibody can be an Fv region comprising a variable light chain (V _L ) and a variable heavy chain (V _H ). The light and heavy chains can be linked directly or via a linker. As used herein, a linker is a conformation that is covalently linked to the light and heavy chains and can specifically bind between the two chains to the epitope to which they are directed. Refers to molecules that provide sufficient spacing and flexibility to achieve Protein linkers are particularly preferred because they can be expressed as unique components of the Ig portion of the fusion polypeptide.

  In another embodiment, recombinantly produced single chain scFv antibodies, preferably humanized scFv, are used in the methods of the invention.

  A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a target molecule, such as VEGF or its receptor. For example, surface labeling and flow cytometric analysis or solid phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with proteins or carbohydrates. See Harlow & Lane (supra) for a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity.

  Examples of antibodies, antibody-related molecules or fragments thereof that can be used in the methods of the present invention include anti-VEGF-A antibodies such as bevacizumab (Avastin) (US Pat. No. 6,054,297), ranibizumab (Lucentis) (US Pat. 6,407,213) and those described in US Pat. No. 5,730,977 and US Patent Application No. 2002032315; anti-VEGF-B antibodies, such as US Patent Application No. 2004005671 and WO 07/140534. Anti-VEGF-C antibodies, such as those described in US Pat. No. 6,403,088; anti-VEGF-D antibodies, such as those described in US Pat. No. 7,097,986; anti-VEGFR-1 antibodies Described in, for example, US Patent Application No. 20030888075 Anti-VEGFR-2 antibodies, such as those described in US Pat. No. 6,344,339, WO 99/40118, and US Patent Application No. 2003176664); and anti-VEGFR-3 antibodies, such as US Although what is described in the patent 6824777 is included, it is not limited to these.

Monoclonal antibodies General methods for producing monoclonal antibodies by hybridomas are well known. Immortal antibody-producing cell lines can be created by cell fusion and also by other techniques, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. A panel of monoclonal antibodies produced against the target epitope can be screened for various properties, i.e., isotype and epitope affinity.

  Animal-derived monoclonal antibodies can be used for both direct and in vitro immunotherapy in vivo. However, for example, when mouse-derived monoclonal antibodies are used as therapeutic agents in humans, patients have been found to produce human anti-mouse antibodies. Therefore, animal-derived monoclonal antibodies are not preferred for therapy, particularly for long-term use. Using established genetic engineering techniques, however, it is possible to produce chimeric or humanized antibodies having an animal-derived part and a human-derived part. The animal can be, for example, a mouse or other rodent such as a rat.

  When the variable region of a chimeric antibody is derived from, for example, a mouse and the constant region is derived from a human, the chimeric antibody is generally less immunogenic than a monoclonal antibody derived from a “pure” mouse. These chimeric antibodies are considered more suitable for therapeutic use if “pure” mouse-derived antibodies are found to be inappropriate.

  Methods for producing chimeric antibodies are available to those skilled in the art. For example, the light and heavy chains can be expressed separately using, for example, an immunoglobulin light chain and an immunoglobulin heavy chain in separate plasmids. These can then be purified and assembled into complete antibodies in vitro; methods for achieving such construction have been described (see, eg, Sun et al., 1986). Such a DNA construct may comprise DNA encoding a functionally rearranged gene for the variable region of an antibody light or heavy chain linked to DNA encoding a human constant region. Hybridomas transfected with DNA constructs for lymphoid cells such as myeloma cells or light and heavy chains can express and construct antibody chains.

  In vitro reaction parameters for the formation of IgG antibodies from shortened isolated light and heavy chains have also been described. Co-expression of the light and heavy chains in the same cell is also possible to achieve intracellular association and ligation of the heavy and light chains to the complete H2L2 IgG antibody. Such co-expression can be achieved using the same or different plasmids in the same host cell.

  In another preferred embodiment of the invention, the antibody is an antibody produced by humanization, ie molecular modeling techniques, in which the human content of the antibody is maximized, eg, the parent rat, Little or no loss of binding affinity due to the variable regions of rabbit or mouse antibodies. The methods described below can be applied to antibody humanization.

  There are several factors to consider when deciding which human antibody sequence to use in humanization. Humanization of light and heavy chains is considered independently of each other, but the basis is basically the same for each.

  This selection process is based on the following rationale: The antigen specificity and affinity of a given antibody is mainly determined by the amino acid sequence of the variable region CDR. The variable domain framework residues contribute little or no directly. The main function of the framework regions is to keep the CDRs in their proper spatial orientation for recognizing antigens. Thus, replacing an animal, for example, a rodent CDR, with a human variable domain framework can be used to determine their proper spatial orientation when the human variable domain framework is highly homologous to the animal variable domain from which it originated. Is very likely to bring about retention. Preferably, therefore, human variable domains that are highly homologous to animal variable domains should be selected. Suitable human antibody variable domain sequences can be selected as follows.

  Step 1. A computer program is used to search all available protein (and DNA) databases for human antibody variable domain sequences that are most homologous to the variable domains of animal-derived antibodies. The output of a suitable program is a list of sequences that are most homologous to the animal-derived antibody, a percent homology to each sequence, and an alignment of each sequence to the animal-derived sequence. This is done independently for both heavy and light chain variable domain sequences. The above analysis is more easily accomplished when only human immunoglobulin sequences are included.

  Step 2. Human antibody variable domain sequences are listed and compared for homology. The comparison is performed primarily on the length of the CDRs, except for the heavy chain CDR3, which is highly variable. Human heavy chains and κ and λ light chains are divided into subgroups: three subgroups of heavy chains, four subgroups of κ chains, and six subgroups of λ chains. The size of the CDR within each subgroup is similar, but varies between subgroups. Usually, as an initial approximation of homology, it is possible to match the CDRs of animal-derived antibodies to one of the human subgroups. Next, antibodies carrying similar lengths of CDRs are compared for amino acid sequence homology, particularly within the CDRs, but also in the surrounding framework regions. The most homologous human variable domain is selected as the framework for humanization.

Actual Humanization Methods / Techniques Antibodies can be humanized by transplanting the desired CDRs into a human framework according to European Patent Application EP-A-0239400. A DNA sequence encoding the desired reconstituted antibody can therefore be generated starting with human DNA that wishes to reconstitute its CDRs. The animal-derived variable domain amino acid sequence containing the desired CDR is compared to that of the selected human antibody variable domain sequence. Residues within the human variable domain are selected that need to be changed to the corresponding residues in the animal to create a human variable region incorporating an animal-derived CDR. There may also be residues that need to be substituted in, added to, or deleted from the human sequence in the human sequence.

  Oligonucleotides are synthesized that can be used to mutagenize the human variable domain framework to include the desired residues. These oligonucleotides can be of any convenient size. Usually only the length is limited by the capabilities of the particular synthesizer available. Methods of oligonucleotide-directed in vitro mutagenesis are well known.

  Alternatively, humanization can be achieved using the recombinant polymerase chain reaction (PCR) of WO 92/07075. Using this method, CDRs can be spliced between framework regions of human antibodies. In general, the technique of WO 92/07075 can be performed using a template containing two human framework regions, AB and CD, and a CDR between them that is to be replaced by a donor CDR. . Primer A and B are used to amplify framework region AB, and primers C and D are used to amplify framework region CD. However, each of primers B and C also contains additional sequences at their 5 ′ end corresponding to all or at least part of the donor CDR sequence. Primers B and C overlap by a length sufficient to anneal their 5 'ends to each other under conditions that allow PCR to be performed. Thus, the amplified regions AB and CD can be spliced by overlap extension to produce a humanized product in a single reaction.

After a mutagenesis reaction that reconstitutes the antibody, the mutated DNA is ligated to the appropriate DNA encoding the light or heavy chain constant region, cloned into an expression vector, and transformed into a host cell, preferably a mammalian cell. Can be transfected. These steps can be performed in a conventional manner. The reconstituted antibody is therefore
(A) at least an Ig heavy or light chain variable domain, a variable domain comprising a framework region from a human antibody, and a DNA sequence encoding a CDR necessary for the humanized antibody of the present invention. Preparing a first replicable expression vector comprising a suitable promoter;
(B) preparing a second replicable expression vector comprising a suitable promoter operably linked to a DNA sequence encoding at least the variable domain of each complementary Ig light or heavy chain;
(C) transforming a cell line with the first or both prepared vectors; and (d) culturing said transformed cell line to produce said altered antibody. .

  Preferably, the DNA sequence of step (a) encodes both the variable domain and the constant domain of each of the human antibody chains. Humanized antibodies can be made using any suitable recombinant expression system. The cell line transformed to produce the altered antibody is an immortalized mammal that is a Chinese hamster ovary (CHO) cell line or conveniently a lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line. It can be an animal cell line. Cell lines can also include normal lymphoid cells, such as B cells, that have been immortalized by transformation with a virus, such as Epstein-Barr virus. Most preferably, the immortalized cell line is a myeloma cell line or a derivative thereof.

CHO cells used for antibody expression may be dihydrofolate reductase (dhfr) deficient and therefore may be thymidine and hypoxanthine dependent for growth. The parental dhfr ^- CHO cell line is transfected with DNA encoding the dhfr gene that allows selection of antibodies and CHO cell transformants of the dhfr positive phenotype. Selection is performed by culturing the colonies in a medium lacking thymidine and hypoxanthine, the absence of thymidine and hypoxanthine causing the non-transformed cells to grow and the transformed cells to reuse the folate pathway, thereby Prevent bypassing the selection system. These transformants usually express a low level of the target DNA by simultaneous integration of the target transfected DNA and the DNA encoding dhfr. The expression level of DNA encoding the antibody can be increased by amplification using methotrexate (MTX). This agent is a direct inhibitor of the enzyme dhfr, allowing the isolation of resistant colonies that amplify their dhfr gene copy number sufficient to survive under these conditions. Since dhfr and the DNA sequence encoding the antibody are closely linked in the original transformant, co-amplification usually occurs, thus increasing the expression of the desired antibody.

  Another preferred expression system for use with CHO or myeloma cells is the glutamine synthetase (GS) amplification system described in WO 87/04462. This system involves transfection of cells with DNA encoding the GS enzyme and DNA encoding the desired antibody. Next, cells that can be presumed to have grown in a glutamine-free medium and thus have incorporated DNA encoding GS are selected. These selected clones are then subjected to inhibition of the GS enzyme using methionine sulfoximine (Msx). In order to survive, the cells amplify DNA encoding GS with simultaneous amplification of DNA encoding the antibody.

  The cell line used to make the humanized antibody is preferably a mammalian cell line, but any other suitable cell line, such as a bacterial cell line or a yeast cell line, can also be used selectively. In particular, it is expected that E. coli-derived bacterial strains can be used. The resulting antibody is tested for functionality. If functionality is lost, it is necessary to return to step (2) to change the framework of the antibody.

  Once expressed, full-length antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be obtained using standard procedures in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, gels. It can be recovered by electrophoresis or the like and purified (generally, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982)). A substantially pure immunoglobulin with a homogeneity of at least about 90-95% is preferred, with a homogeneity of 98-99% or more being most preferred for pharmaceutical use. Once purified, partially or homogeneously as desired, humanized antibodies can then be used in therapy or to develop and perform assay procedures, immunofluorescent staining, etc. (generally Lefkovits and Pernis ( editors), Immunological Methods, VoIs. I and II, Academic Press, (1979 and 1981)).

An antibody with a fully human variable region is also modified to produce such an antibody in response to antigen administration, but the antigen is administered to a transgenic animal in which its endogenous locus has been abolished Can be produced. Various subsequent manipulations can be performed to obtain either the antibody itself or an analog thereof (see, eg, US Pat. No. 6,075,181).
Gene silencing

  In one embodiment, VEGF signaling is disrupted using gene silencing. The terms “RNA interference”, “RNAi” or “gene silencing” generally refer to a double-stranded RNA (dsRNA) molecule in which the double-stranded RNA shares substantial or complete homology. It refers to the process of reducing expression. More recently, however, it has been shown that gene silencing can be achieved using non-RNA duplex molecules (see, eg, US Patent Application No. 200700046667).

  RNA interference (RNAi) is particularly useful for specifically inhibiting the production of specific RNAs and / or proteins. Without wishing to be bound by theory, Waterhouse et al. (1998) provided a model for the mechanism that dsRNA (double stranded RNA) may be used to reduce protein production. This technique is based on the presence of a dsRNA molecule comprising essentially the same sequence as the mRNA of the gene of interest or part thereof, in this case the mRNA encoding the polypeptide according to the invention. Conveniently, the dsRNA can be made from a single promoter in a recombinant vector or host cell, in which the sense and antisense sequences are flanked by irrelevant sequences. This sequence allows the sense and antisense sequences to hybridize to form a dsRNA molecule, with unrelated sequences forming a loop structure. The design and production of suitable dsRNA molecules for the present invention is described in particular by Waterhouse et al. (1998), Smith et al. (2000), WO 99/32619, 99/53050, In view of 99/49029 and 01/34815, it is well within the ability of one skilled in the art.

  The present invention includes the use of nucleic acid molecules comprising and / or encoding double stranded regions for gene silencing. The nucleic acid molecule is typically RNA, but can include DNA, chemically modified nucleotides and non-nucleotides.

  The double stranded region should be at least 19 contiguous nucleotides, eg, about 19-23 nucleotides, or may be longer, eg, 30 or 50 nucleotides, or 100 nucleotides or more. A full-length sequence corresponding to the entire gene transcript can also be used. Preferably they are about 19 to about 23 nucleotides in length.

  The degree of identity of the double-stranded region of the nucleic acid molecule to the targeted transcript should be at least 90%, more preferably 95-100%. The percent identity of a nucleic acid molecule is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty = 5 and a gap extension penalty = 0.3. Preferably, the two sequences are aligned over their entire length.

  Nucleic acid molecules, of course, can include unrelated sequences that can function to stabilize the molecule.

  As used herein, the term “small interfering RNA” or “siRNA” refers to a nucleic acid comprising a ribonucleotide that can inhibit or downregulate gene expression, for example, by mediating RNAi in a sequence-specific manner. Refers to a molecule, wherein the double-stranded portion is less than 50 nucleotides in length, preferably from about 19 to about 23 nucleotides in length. For example, the siRNA can be a nucleic acid molecule that includes self-complementary sense and antisense regions, wherein the antisense region includes a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof, the sense region comprising: It has a nucleotide sequence corresponding to the target nucleic acid sequence or part thereof. siRNA can be constructed from two separate oligonucleotides, one strand is the sense strand and the other is the antisense strand, where the antisense strand and the sense strand are self-complementary.

  As used herein, the term siRNA is another term used to describe a nucleic acid molecule capable of mediating sequence-specific RNAi, such as microRNA (miRNA), small hairpin RNA (shRNA). ), Small interfering oligonucleotides, small interfering nucleic acids (siNA), small interfering modified oligonucleotides, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA) and the like. In addition, as used herein, the term RNAi is equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, or epigenetics. Is intended. For example, the siRNA molecules of the invention can be used to silence genes geneally at both post-transcriptional or pre-transcriptional levels. In a non-limiting example, epigenetic regulation of gene expression by the siRNA molecules of the invention can result from modification of chromatin structure via siRNA to alter gene expression.

  Preferred small interfering RNA (“siRNA”) molecules comprise a nucleotide sequence that is identical to about 19-23 contiguous nucleotides of the target mRNA. In one embodiment, the target mRNA sequence starts at dinucleotide AA and is about 30-70% (preferably 30-60%, more preferably 40-60%, even more preferably about 45% -55%) GC. Does not have a high percent identity to any nucleotide sequence other than the target in the genome of the avian (preferably chicken) into which it is introduced, as determined by, for example, standard BLAST searches .

  By "shRNA" or "Small hairpin RNA", less than about 50 nucleotides, preferably about 19 to about 23 nucleotides are base paired with complementary sequences located on the same RNA molecule, and the sequences complementary to said sequences Means an siRNA molecule that is separated by an unpaired region of at least about 4-15 nucleotides, said unpaired region forming a single stranded loop on a stem structure created by two regions of base complementarity Is done. Examples of single stranded loop sequences are 5'UUCAGAGA3 'and 5'UUUGUGUAG3'.

  The shRNA involved is a dual or bifinger and multi-finger hairpin dsRNA in which the RNA molecule contains two or more such stem loop structures separated by a single stranded spacer region.

  There are well established criteria for designing siRNAs (see, eg, Elbashire et al., 2001; Amarzguioui et al., 2004; Reynolds et al., 2004). Details are found on several commercial suppliers such as Ambion, Dharmacon, GenScript, and OligoEngine websites. Typically, many siRNAs must be made and screened to compare their effectiveness.

  Once designed, dsRNA for use in the methods of the invention can be made by any method known in the art, such as by in vitro transcription, by recombinant methods, or by synthetic means. siRNA can be generated in vitro by using recombinant enzymes such as T7 RNA polymerase and a DNA oligonucleotide template, or can be prepared in vivo, eg, in cultured cells. In a preferred embodiment, the nucleic acid molecule is produced synthetically.

  In addition, a method has been described for generating hairpin siRNA from a vector, including, for example, an RNA polymerase III promoter. Various vectors have been constructed to make hairpin siRNAs in host cells using either H1-RNA or snU6 RNA promoters. The RNA molecules described above (eg, first portion, linking sequence, and second portion) can be operably linked to such a promoter. When transcribed by RNA polymerase III, the first and second parts form the double-stranded stem of the hairpin and the linking sequence forms a loop. The pSuper vector (OligoEngineers Ltd., Seattle, Wash.) can also be used to generate siRNA.

  In order to improve the properties of the nucleic acid molecules of the invention, modified forms or analogues of nucleotides can be introduced. Improved properties include high nuclease resistance and / or high ability to permeate cell membranes. Thus, terms such as “polynucleotide” and “double-stranded RNA molecule” include, for example, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl-adenines, 5- Halouracil, 5-halocytosine, 6-azacytosine and 6-azathymine, pseudouracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyladenine, 8-hydroxyl adenine and other 8-substitutions Adenine, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine and other substituted guanines, other aza and deazaadenine, other aza and deazaguanine, 5-trifluoromethyluracil And 5-trif Such Oroshitoshin, but not limited to, including synthetically modified bases.

  In one embodiment, the ds molecule, preferably dsRNA, comprises any one or more at least 19 contiguous nucleotides of the sequence of nucleotides provided as SEQ ID NOs: 9-16, wherein T is replaced with U. The portion of the molecule that is double-stranded, including the containing oligonucleotide, is at least 19 base pairs in length and contains the oligonucleotide.

  Examples of ds molecules that can be used in the methods of the present invention are: Chinese Patent No. 1804038, Chinese Patent No. 1834254, International Publication No. 08/045576, US Patent Application No. 2006025370, US Patent Application No. 2006094032, British Patent 2,406,569, Canadian Patent No. 2537085, International Publication No. WO 03/070910, U.S. Patent Application Nos. 20060173332, No. 2010522066, No. 2005054596, No. 2004029832, No. 2004038163, No. 2005148530. And those described in JP 2005171039 A, but are not limited thereto.

Antisense polynucleotide The term “antisense polynucleotide” refers to a DNA that encodes a polypeptide of the invention and that is complementary to at least a portion of a specific mRNA molecule capable of interfering with post-transcriptional events, eg, mRNA translation. It is taken to mean a molecule of RNA or a combination thereof. The use of antisense methods is well known in the art (see, eg, G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)). Senior (1998) states that antisense methods are now a very widely established technique for manipulating gene expression.

  The antisense polynucleotide of the invention hybridizes to the target polynucleotide under physiological conditions. As used herein, the term “antisense polynucleotide that hybridizes under physiological conditions” means that the polynucleotide (fully or partially single stranded) is at least a cell, preferably It means that a double-stranded polynucleotide can be formed under normal conditions in human cells with mRNA encoding a protein, such as that provided in any one of SEQ ID NOs: 9-16.

  Antisense molecules can include sequences corresponding to structural genes or sequences that cause control over gene expression or splicing events. For example, the antisense sequence can correspond to a target coding region of a gene of the invention, or a 5 'untranslated region (UTR) or 3'-UTR or a combination thereof. The antisense sequence can be partially complementary to the intron sequence, which can be removed by splicing during or after transcription, preferably only the exon sequence of the target gene. In general, considering these larger UTR differences, targeting these regions provides greater specificity of gene inhibition.

  The length of the antisense sequence should be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, more preferably at least 100, 200, 500 or 1000 nucleotides. A full-length sequence complementary to the entire gene transcript can be used. The length is most preferably 100-2000 nucleotides. The degree of identity of the antisense sequence to the target transcript should be at least 90%, more preferably 95-100%. An antisense RNA molecule can, of course, contain unrelated sequences that can function to stabilize the molecule.

  Examples of antisense polynucleotides that can be used in the methods of the present invention include, but are not limited to, those described in US Patent Application No. 2003186920 and International Publication No. 07/013704.

Catalytic Polynucleotide The term catalytic polynucleotide / nucleic acid specifically recognizes an individual substrate and catalyzes the chemical modification of this substrate, also known as a DNA molecule or DNA-containing molecule (also known in the art as “deoxyribozyme”). ) Or RNA or RNA-containing molecules (also known as “ribozymes”). The nucleobases in the catalytic nucleic acid can be bases A, C, G, T (and U for RNA).

  Typically, a catalytic nucleic acid includes an antisense sequence for specific recognition of a target nucleic acid and an enzymatic activity that cleaves the nucleic acid (also referred to herein as a “catalytic domain”). Particularly useful ribozymes in the present invention are hammerhead ribozymes (Haseloff and Gerlach, 1988; Perriman et al, 1992) and hairpin ribozymes (Shippy et al, 1999).

  Ribozymes and ribozyme-encoding DNAs for use in the present invention can be chemically synthesized using methods well known in the art. Ribozymes can also be prepared from a DNA molecule operatively linked to a promoter for an RNA polymerase promoter, such as T7 RNA polymerase or SP6 RNA polymerase, which yields an RNA molecule after transcription. Accordingly, a nucleic acid molecule encoding the catalytic polynucleotide of the present invention, ie, DNA or cDNA, is also provided by the present invention. If the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, ribozymes can be produced in vitro after incubation with RNA polymerase and nucleotides. In another embodiment, the DNA can be inserted into an expression cassette or transcription cassette. After synthesis, RNA molecules can be modified by ligation to DNA molecules that have the ability to stabilize the ribozyme and make it resistant to RNases.

  With respect to the antisense polynucleotides described herein, the catalytic polynucleotides of the invention are also targeted nucleic acid molecules under “physiological conditions”, ie, intracellular conditions (especially conditions in animal cells, eg, human cells). It should be capable of hybridizing to (eg mRNA encoding any polypeptide provided in SEQ ID NOs: 1-8).

  Examples of ribozymes that can be used in the methods of the present invention include those described in US Pat. No. 6,346,398, Ciafre et al. (2004) and Weng et al. (2005). It is not limited.

Gene Therapy The therapeutic polynucleotides described herein can be used in accordance with the present invention by expression of such polynucleotides in a therapeutic format often referred to as “gene therapy”. Thus, cells from a patient can be manipulated with a polynucleotide, such as DNA or RNA, to encode the polynucleotide ex vivo. The engineered cells can then be provided to a patient to be treated with the polynucleotide or, if applicable, the polypeptide encoded thereby (eg, a VEGF antibody). In this embodiment, cells, such as stem cells or differentiated stem cells, can be manipulated ex vivo, for example by use of retroviral plasmid vectors for transformation. Such methods are well known in the art and their use in the present invention is apparent from the teachings herein.

  In addition, cells can be manipulated in vivo for expression of the polynucleotide in vivo by procedures known in the art. For example, the polynucleotide can be engineered for expression in a replication defective retroviral vector or adenoviral vector or other vector (eg, a poxvirus vector). The expression construct can then be isolated. When a packaging cell is transduced with a plasmid vector containing RNA encoding a polynucleotide described herein, the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells can be administered to a patient for manipulation of the cells in vivo and expression of the polynucleotide in vivo. These and other methods for administering polynucleotides should be apparent to those skilled in the art from the teachings of the present invention.

  The retroviruses from which the above mentioned retroviral plasmid vectors can be derived are Moloney murine leukemia virus, spleen necrosis virus, rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, gibbon leukemia virus, human immunodeficiency virus, adenovirus, bone marrow Including but not limited to proliferative sarcoma virus and breast cancer virus. In a preferred embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus.

  Such vectors contain one or more promoters for expressing the polynucleotide. Suitable promoters that may be used include, but are not limited to, retroviral LTR; SV40 promoter; and human cytomegalovirus (CMV) promoter. Cell promoters such as eukaryotic promoters may also be used, including but not limited to histones, RNA polymerase III, metallothionein promoters, heat shock promoters, albumin promoters, human globin promoters and α-actin promoters. Additional viral promoters that can be used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

Retroviral plasmid vectors can be used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected include PE501, PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17-H2, YCRE, YCRIP, GP + E-86, GP + envAm12, and Miller (1990), including but not limited to the DAN cell line. The vector can transduce the packaging cells via any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO ₄ precipitation. In one alternative method, the retroviral plasmid vector can be encapsulated in a liposome or can be conjugated to a lipid and then administered to a host.

  The producer cell line produces infectious retroviral vector particles containing the polynucleotide. Such retroviral vector particles can then be used to transduce eukaryotic cells in vitro or in vivo. Transduced eukaryotic cells express the polynucleotide and, if applicable, produce the polypeptide encoded thereby. Eukaryotic cells that can be transduced include embryonic stem cells, retinal stem cells, embryonal carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, muscle cells (particularly skeletal muscle cells), endothelial cells and Including but not limited to bronchial epithelial cells.

  In one embodiment, cells administered as part of a combination therapy are not cells that have been genetically modified so that they produce a compound. In particularly preferred embodiments, the cells administered as part of a combination therapy are not cells that have been genetically modified so that they produce anti-VEGF monoclonal antibodies.

  A selectable marker may be included in the construct or vector to observe successful genetic modification and for selection of cells that have incorporated the polynucleotide. Non-limiting examples include drug resistance markers such as G148 or hygromycin. In addition, negative selection can also be used, for example when the marker is the HSV-tk gene. This gene sensitizes cells to agents such as acyclovir and gancyclovir. The NeoR (neomycin / G148 resistance) gene is commonly used, but any convenient marker gene whose gene sequence is not already present in the target cell can be used. Further non-limiting examples include low affinity nerve growth factor (NGFR), enhanced green fluorescent protein (EFGP), dihydrofolate reductase gene (DHFR), bacterial hisD gene, mouse CD24 (HSA), mouse CD8a (lyt) , Bacterial genes conferring resistance to puromycin or phleomycin, and β-galactosidase.

  Additional polynucleotide sequences may be introduced into the cell on the same vector, or may be introduced into the host cell on a second vector. In a preferred embodiment, the selectable marker is contained on the same vector as the polynucleotide.

  The present invention also includes genetically modifying the promoter region of an endogenous gene such that expression of the endogenous gene is upregulated, resulting in increased production of the encoded protein compared to wild type cells. Includes.

  In a useful embodiment of the invention, the cells are genetically modified to contain genes that disrupt or inhibit angiogenesis. The gene may encode a cytotoxic agent, such as lysine. In another embodiment, the gene encodes a cell surface molecule that elicits immune rejection. For example, the cells can be genetically modified to produce α1,3 galactosyltransferase. This enzyme synthesizes the α1,3 galactosyl epitope, the major heterophilic antigen, whose expression is hyperacute in transgenic endothelial cells by preformed circulating antibodies and / or by T cell-mediated immune rejection. Causes immune rejection.

  Gene therapy according to the present invention may involve a transient (temporary) presence of a gene therapy polynucleotide in a patient or a permanent introduction of the polynucleotide into the patient.

Compositions and their administration Typically, the cells and compounds of the invention are administered in a pharmaceutical composition containing at least one pharmaceutically acceptable carrier. Furthermore, one aspect of the invention relates to a composition comprising a compound that disrupts cellular and VEGF signaling, and optionally a pharmaceutically acceptable carrier.

  The term “pharmaceutically acceptable” is used within the scope of sound medical judgment and in contact with human and animal tissues without undue toxicity, irritation, allergic reactions or other problems or complications. Refers to compounds, materials, compositions and / or dosage forms that are suitable for, and commensurate with a reasonable benefit / risk ratio. As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation. Means material.

  Pharmaceutically acceptable carriers include saline, aqueous buffer, solvents and / or dispersion media. The use of such carriers is well known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists. Preferably, the solution is stable under the conditions of manufacture and storage and is protected from the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

  Some examples of materials and solutions that can serve as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene. Glico (12) Esters such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water; 17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer; (21) polyester, polycarbonate and / or polyanhydrides; and (22) others used in pharmaceutical formulations. Of non-toxic compatible substances.

  Pharmaceutical compositions containing cells useful for the methods of the invention can contain a polymeric carrier or an extracellular matrix.

  A variety of biological or synthetic solid matrix materials (ie, solid support matrices, bioadhesives or bandages, and biological / medical scaffolds) are suitable for use in the present invention. The matrix material is preferably medically acceptable for use in in vivo applications. Non-limiting examples of such medically acceptable and / or biologically or physiologically acceptable or compatible materials include solid matrix materials that are absorbable and / or nonabsorbable, For example, small intestine submucosa (SIS), eg, from porcine (and other SIS sources); cross-linked or non-cross-linked alginate, hydrocolloid, foam, collagen gel, collagen sponge, polyglycolic acid (PGA) mesh, polyglactin ( PGL) mesh, fleece, foam bandages, bioadhesives (eg fibrin glue and fibrin gel) and one or more layers of de-epidermized skin equivalents Not.

  Fibrin glue is one class of surgical sealant that has been used in various clinical environments. As those skilled in the art will appreciate, a number of sealants are useful in compositions for use in the methods of the present invention. However, preferred embodiments of the present invention relate to the use of cells and fibrin glue as described herein.

  As used herein, the term “fibrin glue” refers to an insoluble matrix formed by the cross-linking of fibrin polymers in the presence of calcium ions. Fibrin glue may be formed from fibrinogen or a derivative or metabolite thereof derived from a biological tissue or fluid that forms a fibrin matrix, fibrin (soluble monomer or polymer) and / or a complex thereof. Alternatively, fibrin glue can be formed from fibrinogen or a derivative or metabolite thereof, or fibrin produced by recombinant DNA technology.

  Fibrin glue can also be formed by the interaction of fibrinogen with a catalyst for fibrin glue formation (eg thrombin and / or factor XIII). As will be appreciated by those skilled in the art, fibrinogen is cleaved by proteolysis in the presence of a catalyst (eg thrombin) and converted to fibrin monomer. Fibrin monomers can then form polymers that can crosslink to form a fibrin glue matrix. Crosslinking of the fibrin polymer can be facilitated by the presence of a catalyst, such as Factor XIII. The catalyst for fibrin glue formation can be derived from plasma, cryoprecipitates or other plasma fractions containing fibrinogen or thrombin. Alternatively, the catalyst can be produced by recombinant DNA technology.

  The rate at which the clot forms depends on the concentration of thrombin mixed with fibrinogen. Since it is an enzyme-dependent reaction, the higher the temperature (up to 37 ° C.), the faster the clot formation rate. The tensile strength of the clot depends on the concentration of fibrinogen used.

  The use of fibrin glue and methods for its preparation and use are described in US Pat. No. 5,643,192. US Pat. No. 5,643,192 discloses the extraction of fibrinogen and thrombin components from a single donor and the combination of these components alone for use as a fibrin glue. US Pat. No. 5,651,982 describes another method of preparation and use for fibrin glue. US Pat. No. 5,651,982 provides liposomes and fibrin glues for use as topical sealants in mammals.

  Several published documents describe the use of fibrin glue for the delivery of therapeutic agents. For example, US Pat. No. 4,983,393 discloses a composition for use as a vaginal insert comprising agarose, agar, saline, glycosaminoglycan, collagen, fibrin and an enzyme. In addition, US Pat. No. 3,089,815 discloses an injectable pharmaceutical formulation composed of fibrinogen and thrombin, and US Pat. No. 6,468,527 discloses various biological agents for specific sites in the body. And fibrin glues that facilitate delivery of non-biological products. Such a procedure can be used in the method of the invention.

  Suitable polymeric carriers include porous meshes or sponges formed from synthetic or natural polymers, as well as polymer solutions. One form of the matrix is a polymer mesh or sponge; the other form is a polymer hydrogel. Natural polymers that can be used include proteins such as collagen, albumin and fibrin; and polymers of polysaccharides such as alginate and hyaluronic acid. Synthetic polymers include both biodegradable polymers and non-biodegradable polymers. Examples of biodegradable polymers include polymers of hydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA) and polylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, and the like Including a combination of Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, and polyvinyl alcohol.

  Polymers that can form malleable ionically or covalently crosslinked hydrogels are used to encapsulate cells. A hydrogel is a material created when an organic polymer (natural or synthetic) is crosslinked by covalent, ionic or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules to form a gel. is there. Examples of matrices that can be used to form hydrogels include polysaccharides such as alginate, polyphosphazine, and ionically crosslinked polyacrylates, or block copolymers such as Pluronics ™ or each crosslinked by temperature or pH, respectively. Includes Tetronics ™, a polyethylene oxide-polypropylene glycol block copolymer. Other materials include proteins such as fibrin, polymers such as polyvinyl pyrrolidone, hyaluronic acid and collagen.

  In general, these polymers having charged side groups or their monovalent ionic salts are at least partially soluble in aqueous solutions, such as water, buffered saline solutions, or aqueous alcohol solutions. Examples of polymers having acidic side groups that can react with cations are polyphosphazenes, polyacrylic acid, polymethacrylic acid, copolymers of acrylic acid and methacrylic acid, polyvinyl acetate, and sulfonated polymers such as sulfonated polystyrene. Copolymers having acidic side groups formed by reaction of acrylic acid or methacrylic acid with vinyl ether monomers or polymers can also be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups. Examples of polymers having basic side groups that can react with anions are polyvinylamine, polyvinylpyridine, polyvinylimidazole, and some imino substituted polyphosphazenes. Polymer ammonium salts or quaternary salts can also be formed from their backbone nitrogen or pendant imino groups. Examples of basic side groups are amino groups and imino groups.

  Furthermore, the composition used for the method of the present invention may contain at least one other therapeutic agent. For example, the composition may contain an analgesic that helps treat inflammation or pain, another anti-angiogenic compound, or an anti-infective to prevent infection at the site being treated with the composition. More specifically, non-limiting examples of useful therapeutic agents include the following therapeutic categories: analgesics such as non-steroidal anti-inflammatory drugs, opiate agonists and salicylates; anti-infectives such as anthelmintics, Anti-anaerobic bacteria, antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, other β-lactam antibiotics, penicillin antibiotics, quinolone antibiotics Substances, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterial drugs, antituberculosis antimycobacterial drugs, antiprotozoal drugs, antimalarial antiprotozoal drugs, antiviral drugs, antiretroviral drugs, scabicides, anti-inflammatory drugs Drugs, corticosteroid anti-inflammatory drugs, anti-itch / local anesthetics, local anti-infectives, anti-fungal local anti-infectives, anti-viral department Anti-infective drugs; electrolytes and kidney drugs, such as acidifying drugs, alkalizing drugs, diuretics, carbonic anhydrase inhibitors diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolytes Supplements and uric acid excretion drugs; enzymes such as pancreatic enzymes and thrombolytic enzymes; gastrointestinal drugs such as antidiarrheal drugs, gastrointestinal anti-inflammatory drugs, gastrointestinal anti-inflammatory drugs, antacid anti-ulcer drugs, gastric acid pump inhibitors Drugs, gastric mucosal anti-ulcer drugs, H2 blocker anti-ulcer drugs, gallstone dissolution drugs, digestive drugs, emetics, laxatives and fecal softeners, and gastrointestinal motility improvers; general anesthetics such as inhalation anesthetics, halogenated inhalation anesthesia Drugs, intravenous anesthetics, barbituric acid intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics; hormones and hormone regulators such as abortion drugs, adrenal drugs, corticosteroid adrenals Drugs, androgens, antiandrogens, immunobiological agents such as immunoglobulins, immunosuppressants, toxoids and vaccines; local anesthetics such as amide local anesthetics and ester local anesthetics; musculoskeletal drugs such as Anti-gout anti-inflammatory drugs, corticosteroid anti-inflammatory drugs, gold compound anti-inflammatory drugs, immunosuppressive anti-inflammatory drugs, non-steroidal anti-inflammatory drugs (NSAIDs), salicylic acid anti-inflammatory drugs, minerals; and vitamins such as vitamin A , Vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.

  Examples of other anti-angiogenic factors that may be used with the present invention, either as a single composition or as a combination therapy, include platelet factor 4; protamine sulfate; sulfated chitin derivatives (prepared from snow crab shells). Sulfated polysaccharide peptidoglycan complex (SP-PG) (the function of this compound can be enhanced by the presence of steroids such as estrogen and tamoxifen citrate); staurosporine; eg proline analogues, cishydroxyproline, d, L-3,4-dehydroproline, thiaproline, α, α-dipyridyl, regulator of matrix metabolism, including aminopropionitrile fumarate; 4-propyl-5- (4-pyridinyl) -2 (3H) -oxazolone ; Methotrexate; mitoxantrone; heparin; interferon; 2 macros Globulin-serum; ChIMP-3; chymostatin; cyclodextrin tetradecasulfate; eponemycin; camptothecin; fumagillin; sodium gold thiomalate; anti-collagenase serum; α2-antiplasmin; bisantren (National Cancer Institute); (2) -carboxyphenyl-4-chloroanthronylate disodium); thalidomide; angiogenesis-inhibiting steroids; AGM-1470; carboxyaminoimidazole; and metalloproteinase inhibitors such as, but not limited to, BB94 .

  In certain embodiments, the other therapeutic agent can be a growth factor or other molecule that affects cell differentiation and / or proliferation. Growth factors that induce a terminal differentiation state are well known in the art and can be selected from any such factor that has been shown to induce a terminal differentiation state. Growth factors for use in the methods described herein can in certain embodiments be naturally occurring variants or fragments of growth factors.

  Compositions useful for the methods of the invention containing cells result from the subsequent differentiation of cell culture components such as media containing amino acids, metals, coenzyme factors, as well as other subpopulations of cells such as stem cells. It can contain part of what you get.

  Compositions useful for the methods of the invention containing cells can be prepared, for example, by precipitating the cells of interest from a medium and resuspending them in the desired solution or substance. The cells can be sedimented and / or changed from the medium, for example by centrifugation, filtration, ultrafiltration and the like.

  The composition is oral, parenteral, buccal, vaginal, rectal, inhalation, insufflation, sublingual, intramuscular, subcutaneous, topical, intranasal, intraocular, intraperitoneal, intrathoracic, intravenous, hard It can be administered by extramembranous, intrathecal, intraventricular route, and by injection into the joint.

  Cells and / or compounds may be administered to the eye or eyelid using, for example, eye drops, ointments, creams, gels, suspensions, implants, and the like. In another embodiment, ophthalmic disease is treated using intraocular injection. In one embodiment, cells and / or compounds may be administered intravitreally, in another embodiment subretinal, in another embodiment intraretinal, and in another embodiment periocular. obtain. In one embodiment, the cells and / or compounds are placed through a depot coupled to an intraocular lens implant that is inserted during surgery or in an eye that is sutured in the anterior chamber or vitreous. It can be administered via the anterior chamber via the depot into the anterior chamber or vitreous. The cells and / or compounds can be formulated with excipients such as methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidine, neutral polyacrylate (methacrylate) esters, and other viscosity enhancing agents. Cells and / or compounds can be injected into the eye by, for example, subconjunctival or subtenon injection, intravitreal injection, or retrobulbar injection. Cells and / or compounds may be sustained release drug delivery systems such as polymers, matrices, microcapsules, or glycolic acid, lactic acid, glycolic acid and lactic acid combinations alone or in combination with polyethylene glycol, liposomes, silicones, It can be administered by other delivery systems formulated from polyanhydrides, polyvinyl acetate and the like. The delivery device can be implanted in the eye for long-term drug delivery, for example, implanted under the conjunctiva, implanted in the eye wall, or sutured to the sclera. The method of introduction can further be provided by a non-biodegradable device. In particular, the cells and / or compounds can be administered by an implantable lens. The cells and / or compounds can be coated on the lens, dispersed throughout the lens, or both.

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

  Sterile injectable solutions can be prepared by incorporating the required amount of compound and / or cells in a suitable solvent, optionally with one or a combination of the components listed above, followed by filter sterilization. Generally, dispersions are prepared by incorporating the polynucleotide into a sterile vehicle that contains a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, suitable preparation methods include vacuum drying and lyophilization, which pre-sterilize the active ingredient and any additional desired ingredient powders. From those solutions.

  Oral compositions generally contain an inert diluent or an edible carrier. For oral therapeutic administration, the compound or cell can be incorporated with excipients and used in the form of tablets, troches, or capsules, eg, gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain any of the following ingredients or compounds with similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, Disintegrants such as alginic acid, PRIMOGEL or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate or Orange flavor.

  Formulations suitable for nasal administration in which the carrier is a solid include, for example, a coarse powder having a particle size in the range of about 20 to about 500 microns and are drawn from the nose, ie from a container of powder held close to the nose. Administered by rapid inhalation through the nasal passages. Suitable formulations for administration by nebulizer, where the carrier is a liquid, include aqueous or oily solutions of the agent. For administration by inhalation, the compounds or cells can also be delivered in the form of drops or aerosol sprays from a pressurized container or dispenser containing a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer. . Such methods include those described in US Pat. No. 6,468,798.

  Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, surfactants, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays, eye drops, or suppositories. For transdermal administration, the active compound can be formulated in ointments, salves, gels, or creams as generally known in the art. Is done.

One skilled in the art can readily determine the amount of cells, compounds and any carrier (s) administered in the compositions of the invention and in the methods of the invention. In one embodiment, optional additives (in addition to active cells or compounds) are present in phosphate buffered saline in an amount of 0.001-50% (by weight) and the active ingredient is from approximately a few micrograms. On the order of a few milligrams, for example about 0.0001 to about 5% by weight, preferably about 0.0001 to about 1% by weight, even more preferably about 0.0001 to about 0.05% by weight or about 0.001 to about 20%. % By weight, preferably from about 0.01 to about 10% by weight, even more preferably from about 0.05 to about 5% by weight. Needless to say, for any composition administered to an animal or human, and for any particular method of administration, therefore measuring toxicity, eg lethal dose (in a suitable animal model, eg rodent, eg mouse) LD) and LD ₅₀ ; and the dose of the composition (one or more), the concentration of the components therein and the timing of administration of the composition (one or more) that elicits an appropriate response. It is preferable to measure. Such measurements do not require undue experimentation from the knowledge of those skilled in the art, the present disclosure, and the materials cited herein. Also, the time for continuous administration can be confirmed without undue experimentation.

The concentration of cells in the composition of the present invention is at least about 5 × 10 ⁵ cells / mL, at least about 1 × 10 ⁶ cells / mL, at least about 5 × 10 ⁶ cells / mL, at least about 10 ⁷ cells / mL, It can be at least about 2 × 10 ⁷ cells / mL, at least about 3 × 10 ⁷ cells / mL, or at least about 5 × 10 ⁷ cells / mL.

  The compound may be administered in an amount of about 0.001 to 2000 mg / kg body weight per dose, more preferably about 0.01 to 500 mg / kg body weight per dose. Repeated doses can be administered as prescribed by the treating physician.

  The present invention relates to the combined use of cells and compounds that disrupt VEGF signaling to treat or prevent angiogenesis-related diseases. The term “in combination with” or “combination therapy” or variations thereof refers to cells and compounds that are simultaneously, sequentially, or both, either in the same composition or separately (eg, within about 5 minutes of each other). And can be administered sequentially with a time interval of several hours, days or weeks. Such combination therapy can also include one or more administrations. It is contemplated that such combination therapy may involve administering one therapeutic agent multiple times during administration of the other administered agent. The period between doses can range from a few seconds (or less) to hours or hours, such as the nature of the cell or compound (eg, potency, solubility, bioavailability, half-life and kinetic profile), and Depends on patient condition.

  In one embodiment, the compound is administered prior to the cell. This is especially the case when the agent binds to VEGF or its receptor. In one embodiment, the compound is administered about 1 day, 3 days, 5 days, 7 days, 9 days or 14 days before the cells.

  The methods of the invention can be combined with other therapies for treating or preventing ocular diseases and / or angiogenesis related diseases. The nature of these other therapies depends on the specific angiogenesis-related disease. For example, for the treatment or prevention of macular degeneration, the use of the method of the present invention is a method as defined in antioxidants and / or zinc supplements, US Pat. No. 6,942,655. Can be combined with administration of pegaptanib, steroid therapy and / or laser therapy (eg Visudyne ™). For cancer, treatment with the methods of the invention can be combined with surgery, radiation therapy and / or chemotherapy.

  The invention will now be described by the following non-limiting examples and with reference to the accompanying drawings.

A summary of the test plan is shown in FIG.
Materials and Methods Recipient Species Cynomolgus monkey (Macaca fascicularis)
System Cynomolgus
Supplier PCS Preferred Supplier
Age About 1 and a half years to 3 and a half years at the start of treatment Weight range About 2 to 4 kg at the start of treatment
Number of groups 7
Number of animals 6 males / group and 2 spares (44 animals in total)

Rearing facilities Animals were housed in groups (2 or 3) when possible in stainless steel cages with a bar-type floor and automatic water supply valves. Each cage was clearly affixed with a color-coded cage card indicating the project, group, animal number and tattoo.

Each animal was uniquely identified by a permanent skin tattoo. The target conditions for the animal room environment and photoperiod are as follows:
Temperature 24 ± 3 ° C
Humidity 50 ± 20%
Photoperiod 12 hours light and 12 hours dark (except during designated procedures).

Diet All animals receive a standard certified pelleted commercial primate food (2050C Certified Global 20% Protein Primitive Diet: Harlan) twice daily except during designated procedures. I let you. In addition, each animal was given the following supplemental diet daily in any combination: Golden Banana Softy®, Prima-Treat® (5 g format) and / or fresh or dried fruit and environmental enrichment. Prima-Foraging Crumbs® at least once a week as part of the program. After recovery from anesthesia, additional supplemental fruits were given to stimulate appetite and maintain nutrition.

  Maximum allowable concentrations of contaminants in the diet (eg, heavy metals, aflatoxins, organophosphates, chlorinated hydrocarbons, PCBs) were controlled and routinely analyzed by the manufacturer. Freely ingested municipal tap water, softened, purified by reverse osmosis and exposed to ultraviolet light (except during designated procedures). It is believed that there were no known contaminants in the diet that could interfere with the purpose of the test.

Group assignment Prior to initiation of therapy, animals were assigned to treatment groups using computerized randomization procedures with weight stratification as parameters (animals with poor health were also assigned to groups) ( Table 1).

Cell Preparation Monkey bone marrow progenitor cells-cynomolgus monkey (smMPC-cyno) (also referred to as MPC in this example) was prepared as Master Batch Record 3001. Isolated from approximately 15 ml of bone marrow aspirate collected from female cynomolgus monkeys (D.O.B.March 12, 2005) on June 25, 2007 through MES. The bone marrow aspirate suspension was Ficolled and washed to remove anucleated cells (erythrocytes). Nucleated cells were counted and then separated by binding to CA12 antibody (also known as STRO-3 antibody—see WO 2006/108229) and Dynabeads. Cells to which the antibody and beads were bound were positively selected by the magnetic field of the MPC-1 magnet. Positively selected cells were counted and inoculated into T flasks at passage 0 in growth medium. Prior to selection, positive and negative cells were used in the colony formation assay (CFU-F).

  Growth medium was supplied to smmPC-cyno cells. All cultures (passage 0 to pass 5) were fed every 2-4 days until they reached the desired confluency. Subsequently, they were passaged or collected using HBSS wash, then collagenase followed by trypsin / Versen. First passage cells were counted and seeded into T flasks. When the first passage of smMPC-cyno reached the desired confluence, cells were harvested and stored frozen using a controlled rate freezer.

The cryopreserved smMPC-cyno of the first passage was thawed and inoculated into a T flask (second passage). The second passage cell was passaged to Cell Factory at the third passage. The third passage cells were collected and passaged to the Cell Factory to the fourth passage. Excess third passage cells were stored frozen. The fourth passage cell was passaged to 6 × Cell Factory at the fifth passage. When passage 5 smMPC-cyno reached the desired confluence, cells were harvested and stored frozen using a controlled rate freezer. Cells were stored frozen in 50% AlphaMEM, 42.5% Profreeze, and 7.5% DMSO (Tables 2 and 3). Samples were tested for CFU-F assay, FACS, sterility, mycoplasma, and endotoxin (Table 4).

  Cryopreserved smMPC-cyno and human MSC (huMPC) were thawed and inoculated into a differentiation assay optimized for human MPC differentiation along the chondrogenesis, lipogenesis and osteogenic pathways. Lipogenic differentiation and in vitro mineralization were assessed by oil red O and alizarin red staining, respectively.

  Similar to the huMPC counterpart, smMPC was capable of adipogenic differentiation (data not shown). Day 18 cultures of sm and huMPC were stained with Oil Red O for the presence of adipocytes. Both the 1st and 5th subcultures of smMPC retained a large number of lipid-bearing adipocytes when cultured under adipogenic culture conditions.

  After 21 days of osteogenic culture, cells were stained with alizarin red. Osteogenic differentiation was demonstrated by the formation of red stained minerals. Like huMPC, smMPC has the ability to form bone.

Laser-induced choroidal neovascularization Laser-induced choroidal neovascularization (CNV) was performed on the same day as test substance administration. The animals were fasted overnight before the procedure.

  Prior to the procedure, mydriatic eye drops (1% mydriacyl) were applied to both eyes. Animals were given an intramuscular injection of a sedative cocktail of glycopyrrolate, ketamine and xylazine and then anesthetized with isoflurane / oxygen. Under anesthesia, a 9-spot pattern was created around the macula of each eye using an 810 nm diode laser for 0.1 seconds with an initial power setting of 250-300 mW (not created in the macula). If a Bruch's membrane tear was not observed for a particular spot, additional spots were added when deemed appropriate by the veterinary ophthalmologist.

  During the procedure, eye hydration was maintained with saline. Some notable events, such as retinal bleeding, were recorded for each laser spot.

Administration of test substance Lucentis ™ (0.5 mg / mL, 0.3 mL / vial; Novartis Canada) was administered at the time of laser treatment, and MPC plus Lucentis was administered MPC 7 days after laser injury did. A topical ophthalmic antibiotic (gentamicin) was applied twice the day before treatment, immediately after the last injection and twice (am and pm) the day after the injection. Antibiotics were applied after laser treatment if only one injection was performed before laser treatment.

  The conjunctiva is sterilized with U.S. water. S. P. And washed with benzalkonium chloride (Zephiran ™) diluted 1: 10,000 (v / v). A local anesthetic (Proparacaine, 0.5%) was applied to both eyes before and after Zephiran ™. A new syringe was used for each injection using a 30 gauge, 1/2 inch needle. Vehicle, test substance cell suspension and / or 50 μL of Lucentis were administered to both eyes. Immediately after treatment, both eyes were examined (inverted and / or direct fundus examination and / or slit lamp biomicroscopy) and the abnormalities caused by the injection procedure were recorded.

Ocular evaluation Ophthalmic examination Animals were subjected to ophthalmological evaluation on days 2, 14, 26, 34 and 40.

  The mydriatic used was 1% midylacyl. Animals were sedated for examination. Sedative ketamine hydrochloride for injection (Ketamine®), U.S.A. S. P. Were administered by intramuscular injection after an appropriate fasting period.

  The examination is initially performed without a mydriatic (slit lamp only), and performed by a qualified specialist veterinary ophthalmologist, and then repeated after mydriatic drug administration (slit lamp and / or inversion and / or direct Image fundus examination). Fundus photographs were taken before treatment of each animal and when deemed necessary by the animal ophthalmologist during eye examination.

Intraocular pressure measurement After intraocular examination (except for examination immediately after administration), intraocular pressure (IOP) was measured. Prior to measurement, a local anesthetic (alkaine, 0.5%) was instilled. Measurements were performed using Tono-Pen XL ™ or TonoVet. The same instrument type was used throughout the test.

Electroretinogram examination Electroretinogram recording was performed once for all animals and on days 27 and 41 before treatment. Animals were dark-adapted at least 30 minutes prior to ERG recording. Animals were given an intramuscular injection of a sedative cocktail of glycopyrrolate, ketamine and xylazine. Midlyacyl (1%) was applied to each eye approximately 5-10 minutes before the test. The eyelid was pulled with a retractor and contact lens electrodes were positioned on the surface of each eye. Needle electrodes were placed on the skin under each eye (reference) and behind the forehead (ground). Carboxymethylcellulose (1%) eye drops were applied to the inner surface of the contact lens electrode before placing them on the eye.

Each ERG test consisted of the following series of dark-adapted single flash stimuli:
1) -30 dB single flash, average 5 single flashes, flash interval 10 seconds 2) -10 dB single flash, average 5 single flashes, flash interval 15 seconds 3) 0 dB, average 2 A single flash, the interval between flashes is about 120 seconds.

After recording the dark adaptation response, the animals were adapted to a background light of about 25-30 cd / m ² for about 5 minutes, followed by an average of 20 sweeps of 1 Hz photopic white flicker, followed by 20 sweeps of 29 Hz light. Adapted to visual flicker.

Fluorescein angiography (except group 7)
Fluorescein angiograms (FA) were obtained once prior to dosing and on days 15, 28, 35 and 42. After an appropriate fasting period, the animals were given an intravenous injection of propofol and then intubated.

  Midlyacyl (1%) was applied to each eye approximately 5-10 minutes before the test. The eyelid was towed with a retractor. Eye hydration was maintained by frequent washing with saline. Immediate intravenous injection of 1 mL of 10% sodium fluorescein, at which point the right eye filling was recorded in motion picture mode for about 20 seconds. Still images were recorded from both eyes approximately 2 and 10 minutes after fluorescein injection. The order of charges was qualitatively evaluated. Individual laser spots on still images were evaluated semi-quantitatively on a scale of 1-4 for leakage.

Statistical analysis The numerical data (only the main test) obtained from the groups 1 to 4 during the test period were used for the calculation of the group mean and standard deviation. For each parameter of interest (excluding ERG, tonometry and FA), group variances were compared using a rubyn test at a significance level of 0.05. If the difference between group variances was not significant, a one-way analysis of variance (ANOVA) was performed. When ANOVA indicated a significant difference between the mean values (p ≦ 0.05), a group mean comparison was performed between the control group and each treatment group using the Dunnett “t” test.

  Whenever the Rubin test indicated inequality group variance (p ≦ 0.05), the Kruskal-Wallis test was used to compare all study groups. If the Kruskal-Wallis test was significant (p ≦ 0.05), the Dan test was used to assess the significance of the difference between the control group and each test group. Data were evaluated on an individual basis, and group means and standard deviations were calculated as appropriate.

  For each pairwise group comparison of interest, significance was reported at the 0.05, 0.01 and 0.001 levels.

Results FIG. 2A shows anti-VEGF monoclonal antibody (Lucentis 0.5 mg / 50 μl) or low (78,100 cells / 50 μl), medium (312,500 cells / 50 μl) injected into non-human primate eyes after laser photocoagulation. ) Or fluorescein angiography results on day 42 after intravitreal injection of either single dose of allogeneic MPC administered at high (1,250,000 cells / 50 μl) concentration. At 42 days after intravitreal injection, the degree of vascular leakage / neovascularization was comparable and was not significantly different between any groups.

  FIG. 2B shows that additional injection of intravitreal allogeneic MPC at the highest concentration (1,250,000 cells / 50 μl) 7 days after administration of intravitreal Lucentis (0.5 mg / 50 μl) immediately after laser photocoagulation The eyes produced significantly lower mean fluorescein angiogram scores, revealing the synergy of the combination (p = 0.03).

  A fluorescein angiogram (FA) using 10% sodium fluorescein was a still image of each eye recorded approximately 2-5 minutes after administration and was quickly injected intravenously. Angiograms were assessed for leakage on day 42 using a semi-quantitative rating scale of 1-4 for each spot that underwent laser photocoagulation.

  Combined treatment with allogeneic MPC at the highest concentration (1,250,000 cells / 50 μl) 7 days after administration of Lucentis (0.5 mg / 50 μl) immediately after laser photocoagulation (lower panel) at all time points considered (Days 15, 28, 35 and 42) resulted in complete prevention of the most severe form of vascular leakage (grade 4 score), whereas in Lucentis alone group all time points beyond day 15 Many grade 4 severe vascular leaks were observed (FIG. 3).

  When all severe lesions (group 3 or 4 severity lesions) were analyzed, the highest concentration (1,250,000 cells / 50 μl) 7 days after administration of Lucentis (0.5 mg / 50 μl) immediately after laser photocoagulation ) Allogeneic MPC was shown to reduce the severity of vascular leakage at all time points compared to Lucentis alone (FIG. 4). This effect was most significant on day 42 at the end of the study (p = 0.013), indicating a long-term effect of synergy.

  Compared to controls that received intravitreal injection of vehicle alone, Lucentis treatment was found to be superior at day 15 in reducing severe grade 4 vascular leakage (FIG. 5). This effect was progressively lost after day 15, possibly reflecting the short half-life of the antibody. In contrast, the highest dose of allogeneic MPC and Lucentis on day 7 after laser coagulation injury completely prevented any grade 4 severe vascular lesions throughout the 42-day study period. These results indicate that the addition of allogeneic MPC to Lucentis changed the temporary effect of anti-VEGF therapy on vascular leakage to a long lasting effect.

  Allogeneic high-dose MPC combination 7 days after administration of Lucentis after laser-induced photocoagulation injury was associated with the number of low-grade (grade 1) vascular leaks throughout the study period compared to animals receiving control or Lucentis alone. An increase was produced (FIG. 6). This indicates that the combination therapy prevented the progression of low-severity vessels to high-severity vessels, an effect that was observed early and was maintained throughout the study period.

  Histopathological analysis on day 42 revealed that combination therapy significantly reduced the incidence of retinal detachment (p <0.01) compared to each of the other groups tested. (FIG. 7). Retinal detachment was observed in 1 out of 12 animals that received concomitant use of allogeneic MPC at the highest concentration (1,250,000 cells / 50 μl) 7 days after administration of Lucentis (0.5 mg / 50 μl) immediately after laser photocoagulation. Only in In contrast, retinal detachment was seen in 7 out of 12 controls, 8 out of 12 treated with high dose MPC alone, and 7 out of 12 treated with Lucentis alone.

  The highest concentration 7 days after administration of Lucentis (0.5 mg / 50 μl) immediately after laser photocoagulation compared to the high incidence of retinal detachment (37/72, 51%) in all animals subjected to laser photocoagulation (37/72, 51%) Only 1 out of 12 animals (8.5%) that received a combination of allogeneic MPC (1,250,000 cells / 50 μl) developed retinal detachment (p <0.01) (FIG. 8). These results indicate that the combination therapy reduced the risk of retinal detachment associated with laser angiogenesis-induced neovascularization to the baseline level observed with the intravitreal injection procedure alone.

  Those skilled in the art will recognize that many variations and / or modifications may be made to the invention shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. This embodiment should therefore be considered exemplary in all respects and should not be construed as limiting.

  All publications discussed above are incorporated herein in their entirety.

  This application is incorporated herein by reference in its entirety, Australian Patent Application No. 2008903339 filed June 30, 2008 and US Patent Application No. 61/133, filed June 30, 2008, both of which are incorporated herein by reference in their entirety. The priority of 607 is claimed.

Any discussion of materials, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. Any or all of these matters existed prior to the priority date of each claim of this application, so they form part of the prior art foundation or common general knowledge in the relevant field of the invention Should not be construed as an admission that
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Claims (31)

  A method of treating or preventing an eye disease in a subject, comprising administering to the subject i) a cell, and ii) a compound that disrupts vascular endothelial growth factor (VEGF) signaling.   Eye diseases are retinal ischemia, retinal inflammation, retinal edema, retinal detachment, macular hole, traction retinopathy, vitreous hemorrhage, traction macular disease, diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, 2. The method of claim 1 selected from the group consisting of macular degeneration, corneal graft rejection, angiogenic glaucoma, post-lens fibroproliferation and / or rubeosis.   The method according to claim 1, wherein the ocular disease is retinal detachment, diabetic retinopathy, retinopathy of prematurity and / or macular degeneration.   The method according to claim 3, wherein the macular degeneration is atrophic age-related macular degeneration or wet age-related macular degeneration.   A method of treating or preventing an angiogenesis-related disease in a subject, comprising administering to the subject i) a cell, and ii) a compound that disrupts vascular endothelial growth factor (VEGF) signaling.   Angiogenesis-related diseases are angiogenesis-dependent cancer, benign tumor, rheumatoid arthritis, psoriasis, ocular neovascular disease, Osler-Weber syndrome, myocardial neovascularization, plaque neovascularization, telangiectasia, hemophilic arthropathy 6. An angiofibroma, wound granulation, intestinal adhesion, atherosclerosis, scleroderma, hyperplastic scar, cat scratch disease, and Helicobacter pylori ulcer. The method described.   The method according to any one of claims 1 to 6, wherein the cell is a stem cell or a progeny cell thereof.   8. The method of claim 7, wherein the stem cells are obtained from bone marrow or eye.   The method according to claim 7 or 8, wherein the stem cell is a mesenchymal progenitor cell (MPC). The mesenchymal progenitor cell is TNAP ⁺ , STRO-1 ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , CD45 ⁺ , CD146 ⁺ , 3G5 ⁺ or any combination thereof. The method described in 1. 11. The method of claim 10, wherein at least some of the STRO-1 ⁺ cells are STRO-1 ^bri .   The method according to any one of claims 7 to 11, wherein the progeny cells are obtained by culturing mesenchymal progenitor cells in vitro.   13. A method according to any one of claims 1 to 12, wherein the compound binds to vascular endothelial growth factor and / or reduces production of vascular endothelial growth factor.   14. The method according to claim 13, wherein the vascular endothelial growth factor is VEGF-A, VEGF-B, VEGF-C and / or VEGF-D.   15. The method of claim 14, wherein the vascular endothelial growth factor is VEGF-A.   16. A compound that decreases vascular endothelial growth factor production binds to hypoxia-inducible factor 1 (HIF-1) and / or reduces hypoxia-inducible factor 1 production. The method described in 1.   13. A method according to any one of claims 1 to 12, wherein the compound binds to vascular endothelial growth factor receptor and / or reduces production of vascular endothelial growth factor receptor.   18. The method according to claim 17, wherein the vascular endothelial growth factor receptor is selected from VEGFR1, VEGFR2 and / or VEGFR3.   19. The method according to claim 18, wherein the vascular endothelial growth factor receptor is VEGFR1 and / or VEGFR2.   13. A compound according to any of claims 1 to 12, wherein the compound binds to a molecule involved in intracellular signaling induced by vascular endothelial growth factor that binds to a vascular endothelial growth factor receptor and / or reduces production of said molecule. The method according to claim 1.   21. A method according to any one of claims 1 to 20, wherein the compound is a polypeptide.   24. The method of claim 21, wherein the polypeptide is an antibody, an antibody-related molecule, and / or a fragment of any one thereof.   21. A method according to any one of claims 1 to 20, wherein the compound is a polynucleotide.   24. The method of claim 23, wherein the polynucleotide is an antisense polynucleotide, a sense polynucleotide, a catalytic polynucleotide, a double stranded RNA molecule, or a polynucleotide encoding any one or more thereof.   25. A method according to any one of claims 1 to 24, wherein at least some of the cells are genetically modified.   Use of a compound that disrupts cellular and VEGF signaling for the manufacture of a medicament for use in a combination therapy for treating or preventing an eye disease in a subject.   Use of a compound that disrupts cellular and VEGF signaling as a medicament for use in combination therapy to treat or prevent eye disease in a subject.   Use of a compound that disrupts cellular and VEGF signaling for the manufacture of a medicament for use in combination therapy to treat or prevent an angiogenesis related disorder in a subject.   Use of a compound that disrupts cellular and VEGF signaling as an agent for use in combination therapy to treat or prevent an angiogenesis related disorder in a subject.   A composition comprising a compound that disrupts cellular and VEGF signaling, and optionally a pharmaceutically acceptable carrier.   A kit comprising a cell and a compound that disrupts VEGF signaling.

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